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1
Shi, Xiaoli, Sandrine Opi, Adrien Lugari, Audrey Restouin, Thibault Coursindel, Isabelle Parrot, Javier Perez, et al. "Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X." Biochemical Journal 431, no. 1 (September 14, 2010): 93–102. http://dx.doi.org/10.1042/bj20100314.
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SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein–protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix–Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 μM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck–Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.
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Ren, Xiu-Rong, Quan-Sheng Du, Yang-Zhong Huang, Shi-Zhou Ao, Lin Mei, and Wen-Cheng Xiong. "Regulation of Cdc42 Gtpase by Proline-Rich Tyrosine Kinase 2 Interacting with Psgap, a Novel Pleckstrin Homology and Src Homology 3 Domain Containing Rhogap Protein." Journal of Cell Biology 152, no. 5 (March 5, 2001): 971–84. http://dx.doi.org/10.1083/jcb.152.5.971.
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Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.
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HURLSTONE, Adam F. L., Ivan A. OLAVE, Nick BARKER, Mascha van NOORT, and Hans CLEVERS. "Cloning and characterization of hELD/OSA1, a novel BRG1 interacting protein." Biochemical Journal 364, no. 1 (May 8, 2002): 255–64. http://dx.doi.org/10.1042/bj3640255.
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A highly conserved multisubunit enzymic complex, SWI/SNF, participates in the regulation of eukaryote gene expression through its ability to remodel chromatin. While a single component of SWI/SNF, Swi2 or a related protein, can perform this function in vitro, the other components appear to modulate the activity and specificity of the complex in vivo. Here we describe the cloning of hELD/OSA1, a 189KDa human homologue of Drosophila Eld/Osa protein, a constituent of Drosophila SWI/SNF. By comparing conserved peptide sequences in Eld/Osa homologues we define three domains common to all family members. A putative DNA binding domain, or ARID (AT-rich DNA-interacting domain), may function in targetting SWI/SNF to chromatin. Two other domains unique to Eld/Osa proteins, EHD1 and EHD2, map to the C-teminus. We show that EHD2 mediates binding to Brahma-related gene 1 (BRG1), a human homologue of yeast Swi2. EHD1 and EHD2 also appear capable of interacting with each other. Using an antibody raised against EHD2 of hELD/OSA1, we detected Eld/Osa1 in endogenous SWI/SNF complexes derived from mouse brain.
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Eulitz, Stefan, Florian Sauer, Marie-Cecile Pelissier, Prisca Boisguerin, Sibylle Molt, Julia Schuld, Zacharias Orfanos, et al. "Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling." Molecular Biology of the Cell 24, no. 20 (October 15, 2013): 3215–26. http://dx.doi.org/10.1091/mbc.e13-04-0202.
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The Xin actin-binding repeat–containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain–binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat–containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.
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Komla-Soukha, Isabelle, and Camille Sureau. "A Tryptophan-Rich Motif in the Carboxyl Terminus of the Small Envelope Protein of Hepatitis B Virus Is Central to the Assembly of Hepatitis Delta Virus Particles." Journal of Virology 80, no. 10 (May 15, 2006): 4648–55. http://dx.doi.org/10.1128/jvi.80.10.4648-4655.2006.
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ABSTRACT The small hepatitis B virus surface antigen (S-HBsAg) is capable of driving the assembly and secretion of hepatitis delta virus (HDV) particles by interacting with the HDV ribonucleoprotein (RNP). Previously, a specific domain of the S-HBsAg protein carboxyl terminus, including a tryptophan residue at position 196 (W196), was proven essential for HDV maturation (S. Jenna and C. Sureau, J. Virol. 73: 3351-3358, 1999). Mutation of W196 to phenylalanine (W196F) was permissive for HBV subviral particle (SVP) secretion but deleterious to HDV virion assembly. Here, the W196F S-HBsAg deficiency was assigned to a loss of its ability for interaction with the large HDV antigen (L-HDAg), a major component of the RNP. Because the overall S-HBsAg carboxyl terminus is particularly rich in tryptophan, an amino acid frequently involved in protein-protein interactions, site-directed mutagenesis was conducted to investigate the function of the S-HBsAg Trp-rich domain in HDV assembly. Single substitutions of tryptophan between positions 163 and 201 with alanine or phenylalanine were tolerated for SVP secretion, but those affecting W196, W199, and W201 were detrimental for HDV assembly. This was proven to result from a reduced capacity of the mutants for interaction with L-HDAg. In addition, a W196S S-HBsAg mutant, which has been described in HBV strains that arose in a few cases of lamivudine-treated HBV-infected patients, was deficient for HDV assembly as a consequence of its impaired capacity for interacting with L-HDAg. Interestingly, the fact that even the most conservative substitution of phenylalanine for tryptophan at positions 196, 199, or 201 was sufficient to ablate interaction of S-HBsAg with L-HDAg suggests that W196, W199, and W201 are located at a binding interface that is central to HDV maturation.
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Gao, Weiqiang, Patricia J. Anderson, Elaine M. Majerus, Elodee A. Tuley, and J. Evan Sadler. "The C-Terminal α-Helix of von Willebrand Factor Domain A2 Interacts with ADAMTS13 C-Terminal Domains To Regulate Substrate Cleavage." Blood 106, no. 11 (November 16, 2005): 410. http://dx.doi.org/10.1182/blood.v106.11.410.410.
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Abstract ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and <10% of VWF64, whereas MDTC cleaved 40% of VWF73 and 30% of VWF64. ADAMTS13 truncated after the first TSP1 repeat (MDT) or the disintegrin domain (MD) had markedly reduced activity, but with prolonged incubation (24 h) at increased concentration (30 nM) both enzymes cleaved most of VWF64 and VWF73 at the expected site. No aberrant cleavage products were detected by Western blotting. The metalloproteinase domain alone (M) was inactive. The selective effects of deleting the cysteine-rich or spacer domain suggest that the C-terminal α-helix of the VWF A2 domain is not essential but facilitates substrate recognition by interacting with specific C-terminal domains of ADAMTS13, particularly the cysteine-rich and spacer domains.
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Zhou, Xi, Jiali Si, Joe Corvera, Gary E. Gallick, and Jian Kuang. "Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins." Biochemical Journal 432, no. 3 (November 25, 2010): 525–38. http://dx.doi.org/10.1042/bj20100862.
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The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)–ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.
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Shepard, Jeremiah, Martin Reick, Sara Olson, and Brenton R. Graveley. "Characterization of U2AF6, a Splicing Factor Related to U2AF35." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 221–30. http://dx.doi.org/10.1128/mcb.22.1.221-230.2002.
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ABSTRACT The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. U2AF35 has multiple functions in pre-mRNA splicing. First, U2AF35 has been shown to function by directly interacting with the AG at the 3′ splice site. Second, U2AF35 is thought to play a role in the recruitment of U2AF65 by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF35 and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF35, designated U2AF26. The N-terminal 187 amino acids of U2AF35 and U2AF26 are nearly identical. However, the C-terminal domain of U2AF26 lacks many characteristics of the U2AF35 RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF26 can associate with U2AF65 and can functionally substitute for U2AF35 in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF26 functions by enhancing the binding of U2AF65 to weak 3′ splice sites. These studies identify U2AF26 as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF35, U2AF26 may play a role in regulating alternative splicing.
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Ma, Liqun, Ke Cheng, Jinyan Li, Zhiqi Deng, Chunjiao Zhang, and Hongliang Zhu. "Roles of Plant Glycine-Rich RNA-Binding Proteins in Development and Stress Responses." International Journal of Molecular Sciences 22, no. 11 (May 29, 2021): 5849. http://dx.doi.org/10.3390/ijms22115849.
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In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.
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DeJournett, Robert E., Ryuji Kobayashi, Shujuan Pan, Chuanfen Wu, Laurence D. Etkin, Richard B. Clark, Oliver Bögler, and Jian Kuang. "Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins." Biochemical Journal 401, no. 2 (December 21, 2006): 521–31. http://dx.doi.org/10.1042/bj20061287.
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The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (α-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.
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Hu, Miaoqing, Luqin Li, Jianbing Chao, Yaqin Zhao, Zhiyun Zhang, and Aihua Liang. "The acidic ribosomal protein P2 from Euplotes octocarinatus is phosphorylated at its N-terminal domain." Biochemistry and Cell Biology 92, no. 1 (February 2014): 23–32. http://dx.doi.org/10.1139/bcb-2013-0063.
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The eukaryotic acid ribosomal P0, P1, and P2 proteins share a conserved flexible C-terminal tail that is rich in acidic residues, which are involved in the interaction with elongation factor 2 during protein synthesis. Our previous work suggested that the acidic ribosomal P proteins from Euplotes octocarinatus have a special C-terminal domain. To further understand this characteristic feature, both P2 and elongation factor 2 from E. octocarinatus were overexpressed, for the first time, in Escherichia coli in this study. GST pull-down assay indicated that P2 protein from E. octocarinatus (EoP2) interacted specifically with the N-terminal domain of elongation factor 2 from E. octocarinatus (EoEF-2) in vitro. The interacting part of EoP2 is in the C-terminal domains, consistent with the observation in other organisms. Phosphorylation of the recombinant EoP2 was performed in vitro using multiple methods such as 31P-NMR spectroscopy, native PAGE, and Phos-tagTM SDS-PAGE. Results showed that ribosomal protein EoP2 was phosphorylated by casein kinase II at serine 21 located at the N terminus. This phosphorylation site identified in EoP2 is quite different from that of P2 from other organisms, in which the phosphorylation site is located in the conserved C-terminal region.
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Li, Yang, Wei Xi, Jianfeng Hao, Li Zhang, Xingpeng Wen, Zhiguo Wu, and Yuxian Zhu. "A Novel Tandem Zinc Finger Protein in Gossypium hirsutum, GhTZF2, Interacts with GhMORF8 to Regulate Cotton Fiber Cell Development." Agronomy 13, no. 2 (February 11, 2023): 519. http://dx.doi.org/10.3390/agronomy13020519.
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Arginine-rich Tandem Zinc Finger (RR-TZF) proteins make up a plant-specific superfamily that participates in plant development, while their roles in cotton fiber development remain to be explored. In this study, we identified an RR-TZF protein-coding gene, GhTZF2, containing two CCCH domains (C-X7-C-X5-C-X3-H-X16-C-X5-C-X4-C-X3-H) and one RR domain at the N-terminus of the two CCCH domains, by comparing the differences of chromatin H3K4me3 modifications between wild-type upland cotton (WT) and the fuzzless-lintless mutant (fl) ovules. GhTZF2 was highly expressed in ovule cells near anthesis, and multiple experiments revealed that GhTZF2 could interact directly with GhMORF8. Homozygotic GhTZF2-knockout cotton lines produced significantly shorter fibers with thinner cell walls. Additionally, comparative transcriptome analysis confirmed that many differentially expressed transcripts contain adenine- and uridine-rich (AU-rich) elements (AREs) in their 3’ untranslated regions (UTR). Together, this study indicated that GhTZF2 may regulate cotton fiber cell development through interacting with GhMORF8, or may be involved in mRNA turnover.
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He, Fan, Wade Borcherds, Tanjing Song, Xi Wei, Mousumi Das, Lihong Chen, Gary W. Daughdrill, and Jiandong Chen. "Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding." Proceedings of the National Academy of Sciences 116, no. 18 (April 15, 2019): 8859–68. http://dx.doi.org/10.1073/pnas.1903077116.
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The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT–DBD interaction.
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Li, He, Lawrence M. Schopfer, Patrick Masson, and Oksana Lockridge. "Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers." Biochemical Journal 411, no. 2 (March 27, 2008): 425–32. http://dx.doi.org/10.1042/bj20071551.
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BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t½) of 11–14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.
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Schmid, Susanne I., and Patrick Hearing. "Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains." Journal of Virology 72, no. 8 (August 1, 1998): 6339–47. http://dx.doi.org/10.1128/jvi.72.8.6339-6347.1998.
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ABSTRACT Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of acis-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AI, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respectivecis-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting,trans-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.
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Haikonen, Tuuli, Minna-Liisa Rajamäki, and Jari P. T. Valkonen. "Interaction of the Microtubule-Associated Host Protein HIP2 with Viral Helper Component Proteinase Is Important in Infection with Potato virus A." Molecular Plant-Microbe Interactions® 26, no. 7 (July 2013): 734–44. http://dx.doi.org/10.1094/mpmi-01-13-0023-r.
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Microtubules (MT) outline and maintain the overall shape of cells and can reorganize cellular membranes to serve as sites of RNA virus replication. Here, we provide data on involvement of an MT-associated protein in infection of plants with a potyvirus, Potato virus A (PVA), representing the largest family of plant-infecting RNA viruses. Our results showed that helper-component proteinase (HCpro)-interacting protein 2 (HIP2) of potato (Solanum tuberosum) is an MT-associated protein similar to Arabidopsis SPR2. Virus-induced silencing of HIP2 in Nicotiana benthamiana resulted in a spiral-like growth phenotype, similar to the Arabidopsis spr2 mutant, and the spr2 phenotype in Arabidopsis was complemented with potato HIP2. HCpro of PVA interacted with HIP2 of potato and tobacco (Nicotiana tabacum). The interaction was detected by bimolecular fluorescence complementation in PVA-infected leaves on MT and MT intersections at the cell cortex. HIP2-HCpro interaction was determined by the C-proximal α-helix-rich domain of HIP2, whereas the N-proximal putative TOG domain and the central coiled-coil domain of HIP2 controlled HIP2 dimerization and binding to MT. Accumulation of PVA was significantly reduced in the HIP2-silenced leaves of N. benthamiana, which indicates that HIP2-HCpro interactions are important for virus infection.
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Abou-Zeid, Nancy, Rudy Pandjaitan, Lucie Sengmanivong, Violaine David, Gwenaelle Le Pavec, Jean Salamero, and Ahmed Zahraoui. "MICAL-like1 mediates epidermal growth factor receptor endocytosis." Molecular Biology of the Cell 22, no. 18 (September 15, 2011): 3431–41. http://dx.doi.org/10.1091/mbc.e11-01-0030.
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Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein–Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.
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Bennett, Erin M., Andrew M. L. Lever, and Jane F. Allen. "Human Immunodeficiency Virus Type 2 Gag Interacts Specifically with PRP4, a Serine-Threonine Kinase, and Inhibits Phosphorylation of Splicing Factor SF2." Journal of Virology 78, no. 20 (October 15, 2004): 11303–12. http://dx.doi.org/10.1128/jvi.78.20.11303-11312.2004.
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ABSTRACT Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase. Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays. The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag. PRP4 is not incorporated into virus particles. HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2. This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1. Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses. We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2. At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.
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Luo, Shuo, Yu Chen, Kwok-On Lai, Juan Carlos Arévalo, Stanley C. Froehner, Marvin E. Adams, Moses V. Chao, and Nancy Y. Ip. "α-Syntrophin regulates ARMS localization at the neuromuscular junction and enhances EphA4 signaling in an ARMS-dependent manner." Journal of Cell Biology 169, no. 5 (June 6, 2005): 813–24. http://dx.doi.org/10.1083/jcb.200412008.
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EphA4 signaling has recently been implicated in the regulation of synapse formation and plasticity. In this study, we show that ankyrin repeat-rich membrane spanning (ARMS; also known as a kinase D–interacting substrate of 220 kD), a substrate for ephrin and neurotrophin receptors, was expressed in developing muscle and was concentrated at the neuromuscular junction (NMJ). Using yeast two-hybrid screening, we identified a PDZ (PSD-95, Dlg, ZO-1) domain protein, α-syntrophin, as an ARMS-interacting protein in muscle. Overexpression of α-syntrophin induced ARMS clustering in a PDZ domain–dependent manner. Coexpression of ARMS enhanced EphA4 signaling, which was further augmented by the presence of α-syntrophin. Moreover, the ephrin-A1–induced tyrosine phosphorylation of EphA4 was reduced in C2C12 myotubes after the blockade of ARMS and α-syntrophin expression by RNA interference. Finally, α-syntrophin–null mice exhibited a disrupted localization of ARMS and EphA4 at the NMJ and a reduced expression of ARMS in muscle. Altogether, our findings suggest that ARMS may play an important role in regulating postsynaptic signal transduction through the syntrophin-mediated localization of receptor tyrosine kinases such as EphA4.
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Zhang, Jin-San, Martin C. Moncrieffe, Joanna Kaczynski, Volker Ellenrieder, Franklyn G. Prendergast, and Raul Urrutia. "A Conserved α-Helical Motif Mediates the Interaction of Sp1-Like Transcriptional Repressors with the Corepressor mSin3A." Molecular and Cellular Biology 21, no. 15 (August 1, 2001): 5041–49. http://dx.doi.org/10.1128/mcb.21.15.5041-5049.2001.
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ABSTRACT Sp1-like proteins are defined by three highly homologous C2H2 zinc finger motifs that bind GC-rich sequences found in the promoters of a large number of genes essential for mammalian cell homeostasis. Here we report that TIEG2, a transforming growth factor β-inducible Sp1-like protein with antiproliferative functions, represses transcription through recruitment of the mSin3A-histone deacetylase complex. The interaction of TIEG2 with mSin3A is mediated by an alpha-helical repression motif (α-HRM) located within the repression domain (R1) of TIEG2. This α-HRM specifically associates with the second paired amphipathic helix (PAH2) domain of mSin3A. Mutations in the TIEG2 α-HRM domain that disrupt its helical structure abolish its ability to both bind mSin3A and repress transcription. Interestingly, the α-HRM is conserved in both the TIEG (TIEG1 and TIEG2) and BTEB (BTEB1, BTEB3, and BTEB4) subfamilies of Sp1-like proteins. The α-HRM from these proteins also mediates direct interaction with mSin3A and represses transcription. Surprisingly, we found that the α-HRM of the Sp1-like proteins characterized here exhibits structural and functional resemblance to the Sin3A-interacting domain previously described for the basic helix-loop-helix protein Mad1. Thus, our study defines a mechanism of transcriptional repression via the interactions of the α-HRM with the Sin3-histone deacetylase complex that is utilized by at least five Sp1-like transcriptional factors. More importantly, we demonstrate that a helical repression motif which mediates Sin3 interaction is not an exclusive structural and functional characteristic of the Mad1 subfamily but rather has a wider functional impact on transcriptional repression than previously demonstrated.
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Nerusheva, Olga O., and Bungo Akiyoshi. "Divergent polo box domains underpin the unique kinetoplastid kinetochore." Open Biology 6, no. 3 (March 2016): 150206. http://dx.doi.org/10.1098/rsob.150206.
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Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1–19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here, we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei . KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region, and all three proteins have weak but significant similarity to the polo box domain (PBD) of PLK. These divergent PBDs of KKT2 and KKT20 are sufficient for kinetochore localization in vivo . We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids.
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Kalthoff, Christoph, Stephanie Groos, Rüdiger Kohl, Stefan Mahrhold, and Ernst J. Ungewickell. "Clint: A Novel Clathrin-binding ENTH-Domain Protein at the Golgi." Molecular Biology of the Cell 13, no. 11 (November 2002): 4060–73. http://dx.doi.org/10.1091/mbc.e02-03-0171.
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We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the γ-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif 423LFDLM, with the γ-adaptin ear homology domain of Golgi-localizing, γ-adaptin ear homology domain 2, with the appendage domain of β2-adaptin and to a lesser extent with the appendage domain of α-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met427-Met605), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of thetrans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors.
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Doliana, Roberto, Simonetta Bot, Gabriella Mungiguerra, Anna Canton, Stefano Paron Cilli, and Alfonso Colombatti. "Isolation and Characterization of EMILIN-2, a New Component of the Growing EMILINs Family and a Member of the EMI Domain-containing Superfamily." Journal of Biological Chemistry 276, no. 15 (January 16, 2001): 12003–11. http://dx.doi.org/10.1074/jbc.m011591200.
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EMILIN (elastinmicrofibrilinterfaselocated Protein) is an elastic fiber-associated glycoprotein consisting of a self-interacting globular C1q domain at the C terminus, a short collagenous stalk, an extended region of potential coiled-coil structure, and an N-terminal cysteine-rich domain (EMI domain). Using the globular C1q domain as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a novel protein. Determination of the entire primary structure demonstrated that this EMILIN-binding polypeptide is highly homologous to EMILIN. The domain organization is superimposable, one important difference being a proline-rich (41%) segment of 56 residues between the potential coiled-coil region and the collagenous domain absent in EMILIN. The entire gene (localized on chromosome 18p11.3) was isolated from a BAC clone, and it is structurally almost identical to that of EMILIN (8 exons, 7 introns with identical phases at the exon/intron boundaries) but much larger (about 40versus8 kilobases) than that of EMILIN. Given these findings we propose to name the novel protein EMILIN-2 and the prototype member of this family EMILIN-1 (formerly EMILIN). The mRNA expression of EMILIN-2 is more restricted compared with that of EMILIN-1; highest levels are present in fetal heart and adult lung, whereas, differently from EMILIN-1, adult aorta, small intestine, and appendix show very low expression, and adult uterus and fetal kidney are negative. Finally, the EMILIN-2 protein is secreted extracellularly byin vitro-grown cells, and in accordance with the partial coexpression in fetal and adult tissues, the two proteins shown extensive but not absolute immunocolocalizationin vitro.
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Li, Youjun, Kenneth Rogulski, Quansheng Zhou, Peter J. Sims, and Edward V. Prochownik. "The Negative c-Myc Target Onzin Affects Proliferation and Apoptosis via Its Obligate Interaction with Phospholipid Scramblase I." Molecular and Cellular Biology 26, no. 9 (May 1, 2006): 3401–13. http://dx.doi.org/10.1128/mcb.26.9.3401-3413.2006.
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ABSTRACT Onzin, the product of a negatively c-Myc-regulated target gene, is highly expressed in myeloid cells. As a result of its interaction with and activation of Akt1 and Mdm2, onzin down-regulates p53. The apoptotic sensitivity of several cell lines is thus directly related to onzin levels. We have conducted a search for additional onzin-interacting proteins and identified phospholipid scramblase 1 (PLSCR1), an endofacial membrane protein, which is proposed to mediate the bidirectional movement of plasma membrane phospholipids during proliferation and apoptosis. PLSCR1 interacts with the same cysteine-rich domain of onzin as do Akt1 and Mdm2, whereas the onzin-interacting domain of PLSCR1 centers around, but does not require, a previously identified palmitoylation signal. Depletion of endogenous PLSCR1 in myeloid cells leads to a phenotype that mimics that of onzin overexpression, providing evidence that PLSCR1 is a physiologic regulator of onzin. In contrast, PLSCR1 overexpression in fibroblasts, which normally do not express onzin, affects neither growth nor apoptosis unless onzin is coexpressed, in which case PLSCR1 completely abrogates onzin's positive effects on proliferation and survival. These findings demonstrate a functional interdependence between onzin and PLSCR1. They further suggest a contiguous link between the earliest events mediated by c-Myc and the latest ones, which culminate at the cell surface and lead to phospholipid reshuffling and cell death.
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Hoque, Mainul, Tara M. Young, Chee-Gun Lee, Ginette Serrero, Michael B. Mathews, and Tsafi Pe'ery. "The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription." Molecular and Cellular Biology 23, no. 5 (March 1, 2003): 1688–702. http://dx.doi.org/10.1128/mcb.23.5.1688-1702.2003.
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ABSTRACT Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.
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El Kasmi, Farid, Eui-Hwan Chung, Ryan G. Anderson, Jinyue Li, Li Wan, Timothy K. Eitas, Zhiyong Gao, and Jeffery L. Dangl. "Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein." Proceedings of the National Academy of Sciences 114, no. 35 (August 14, 2017): E7385—E7394. http://dx.doi.org/10.1073/pnas.1708288114.
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Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogen-derived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana. We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1–RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM.
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Bou Zeidan, Marc, Lourdes Carmona, Severino Zara, and Jose F. Marcos. "FLO11Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide." Applied and Environmental Microbiology 79, no. 19 (July 26, 2013): 6023–32. http://dx.doi.org/10.1128/aem.01647-13.
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ABSTRACTSaccharomyces cerevisiae“flor” yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends onFLO11gene length and expression.FLO11encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm ofS. cerevisiaeflor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functionalFLO11but was not accompanied by increasedFLO11expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that aflo11gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that theFLO11gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.
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Khatlani, Tanvir, Subhashree Pradhan, Vladimir Buchman, and K. Vinod Vijayan. "CIN85 Is a New Protein Phosphatase 2Ac Interacting Protein That Regulates αIIbβ3 Adhesion to Fibrinogen." Blood 120, no. 21 (November 16, 2012): 95. http://dx.doi.org/10.1182/blood.v120.21.95.95.
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Abstract Abstract 95 Platelet activation at the site of injury is dependent on signal transduction events that are mediated by protein kinases and protein phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector (cytoskeletal, signaling and adaptor) proteins are critical for propagating signaling downstream of platelet receptors. Several studies have indicated a key role for protein kinases and their effectors in regulating the functions of integrin αIIbβ3. In contrast, much less is known about the contribution of serine/threonine phosphatases in integrin function, and the identities of effectors regulated by phosphatases are unknown. In this context, we have previously noticed that depletion of the catalytic subunit of protein phosphatase 2A (PP2Ac) enhanced Src activation and augmented αIIbβ3 adhesiveness to immobilized fibrinogen. Since protein-protein interactions form the foundation of cell signaling networks, we sought to identify the potential effectors of PP2Ac. We employed yeast two-hybrid interaction studies with the full length PP2Ac fused to GAL4 binding domain as bait and screened human bone marrow library. A novel interaction of PP2Ac with a protein called CIN85 was identified. Although CIN85 associates with several proteins, an interaction with PP2Ac has not been reported in any cell types. CIN85 (Cbl-interacting protein of 85 kDa) also known as Ruk or SETA is an adaptor protein with three SH3 domains, followed by a proline rich region, a serine rich region and a coiled-coil region. CIN85 participates in vesicle mediated transport and cytoskeleton remodeling. Co-immunoprecipitation (co-IP) experiments validated the interaction of the HA tagged PP2Ac with FLAG tagged CIN85 in 293 cells expressing PP2Ac-HA and CIN85-FLAG. Purified PP2Ac bound to recombinant CIN85-GST protein but not to GST protein, indicating that the in vitro interaction of PP2Ac with CIN85 was direct. Transfection and co-IP experiments with several FLAG tagged truncation mutants of CIN85 in 293 cells revealed that the interaction of PP2Ac with CIN85 was mediated by the proline rich region of CIN85. These studies established a direct interaction of PP2Ac with CIN85. Importantly, the interaction of purified PP2Ac with recombinant CIN85 decreased PP2Ac activity, suggesting that this complex has signaling consequence in vitro. We explored and showed for the first time that CIN85 is expressed in platelets. More importantly, PP2Ac co-immunoprecipitated with CIN85 in human platelets and in 293 αIIbβ3 cells suspended over BSA substrate. Interestingly, adhesion of platelets and 293 αIIbβ3 cells to immobilized fibrinogen induced dissociation of this complex. These studies suggest that the dissociation of PP2Ac-CIN85 complex following integrin stimulation enables CIN85 to propagate outside-in signals by efficiently engaging with other downstream effectors. Consistent with this notion, siRNA mediated depletion of CIN85 significantly (p<0.001) decreased adhesion of 293 αIIbβ3 cells to immobilized fibrinogen. These studies reveal that platelet activation events involve the coupling of the integrin αIIbβ3 adhesion initiated signaling with the phosphatase effector CIN85. Disclosures: No relevant conflicts of interest to declare.
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Melkumov, Georgy. "Recent results of strong interaction program from NA61/SHINE experiment at CERN SPS." EPJ Web of Conferences 204 (2019): 01010. http://dx.doi.org/10.1051/epjconf/201920401010.
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The NA61/SHINE experiment at the CERN SPS pursues a rich program of strong interactions. The main physics goals of this program are the study of properties of the onset of deconfinement and search for the signatures of the critical point of strongly interacting matter by performing the two-dimensional scan in a broad region of energy (beam momentum 13A-158A GeV/c) and system size (p+p, Be+Be, Ar+Sc and Xe+La). Recent NA61/SHINE results on particle spectra and event-by-event fluctuations in p+p, Be+Be and Ar+Sc collisions are shown with emphasis on the measurements of particle ratios, namely of pion a strangeness production, multiplicity fluctuations versus energy and the system size of colliding nuclei. It will be shown that the hadron production properties in heavy ion collisions which change rapidly in the low SPS energy domain and are interpreted as the beginning of quark-gluon plasma production – onset of deconfinement could be also the case in inelastic p+p interactions and probably in Be+Be collisions. The paper presents a selection of NA61/SHINE results on particle production properties discussed together with existing data from the NA49 collaboration. The evolution of non-monotonic structures in the pion and strangeness production as a function of the system size and energy is addressed. The rapid change of hadron production properties from p+p and Be+Be up to Ar+Sc and Pb+Pb collisions can be interpreted as the beginning of the large clusters formation of strongly interacting matter - the onset of fireball. The NA61/SHINE strong interaction programme is presented including the recent status on proton intermittency analysis and strongly intensive fluctuation observables of particle multiplicity and transverse momentum.
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Bazin, Alexandre, Mickaël Cherrier, and Laurent Terradot. "Structural insights into DNA replication initiation in Helicobacter pylori." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1632. http://dx.doi.org/10.1107/s2053273314083673.
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In Gram-negative bacteria, opening of DNA double strand during replication is performed by the replicative helicase DnaB. This protein allows for replication fork elongation by unwinding DNA and interacting with DnaG primase. DnaB is composed of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a flexible linker. The protein forms two-tiered hexamers composed of a NTD-ring and a CTD-ring. In Escherichia coli, the initiator protein DnaA binds to the origin of replication oriC and induces the opening of a AT-rich region. The replicative helicase DnaB is then loaded onto single stranded DNA by interacting with DnaA and with the AAA+ helicase loader DnaC. However, AAA+ loaders are absent in 80% of the bacterial genome, raising the question of how helicases are loaded in these bacteria [1]. In the genome of human pathogen Helicobacter pylori, no AAA+ loader has been identified. Moreover H. pylori DnaB (HpDnaB) has the ability to support replication of an otherwise unviable E. coli strain that bears a defective copy of DnaC by complementation [2]. In order to better understand the properties of HpDnaB we have first shown that HpDnaB forms double hexamers by negative stain electron microscopy [3]. Then, we have then solved the crystal structure of HpDnaB at a resolution of 6.7Å by X-ray crystallography with Rfree/Rfactor of 0.29/0.25. The structure reveals that the protein adopts a new dodecameric arrangement generated by crystallographic three fold symmetry. When compared to hexameric DnaBs, the hexamer of HpDnaB displays an original combination of NTD-ring and CTD-ring symmetries, intermediate between apo and ADP-bound structure. Biochemistry studies of HpDnaB interaction with HpDnaG-CTD and ssDNA provides mechanistic insights into the initial steps of DNA replication in H. pylori. Our results offer an alternative solution of helicase loading and DNA replication initiation in H. pylori and possibly other bacteria that do not employ helicase loaders.
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Balaban, Can, Martin Sztacho, Ludovica Antiga, Ana Miladinović, Masahiko Harata, and Pavel Hozák. "PIP2-Effector Protein MPRIP Regulates RNA Polymerase II Condensation and Transcription." Biomolecules 13, no. 3 (February 24, 2023): 426. http://dx.doi.org/10.3390/biom13030426.
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The specific post-translational modifications of the C-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (RNAPII) correlate with different stages of transcription. The phosphorylation of the Ser5 residues of this domain associates with the initiation condensates, which are formed through liquid-liquid phase separation (LLPS). The subsequent Tyr1 phosphorylation of the CTD peaks at the promoter-proximal region and is involved in the pause-release of RNAPII. By implementing super-resolution microscopy techniques, we previously reported that the nuclear Phosphatidylinositol 4,5-bisphosphate (PIP2) associates with the Ser5-phosphorylated-RNAPII complex and facilitates the RNAPII transcription. In this study, we identified Myosin Phosphatase Rho-Interacting Protein (MPRIP) as a novel regulator of the RNAPII transcription that recruits Tyr1-phosphorylated CTD (Tyr1P-CTD) to nuclear PIP2-containing structures. The depletion of MPRIP increases the number of the initiation condensates, indicating a defect in the transcription. We hypothesize that MPRIP regulates the condensation and transcription through affecting the association of the RNAPII complex with nuclear PIP2-rich structures. The identification of Tyr1P-CTD as an interactor of PIP2 and MPRIP further points to a regulatory role in RNAPII pause-release, where the susceptibility of the transcriptional complex to leave the initiation condensate depends on its association with nuclear PIP2-rich structures. Moreover, the N-terminal domain of MPRIP, which is responsible for the interaction with the Tyr1P-CTD, contains an F-actin binding region that offers an explanation of how nuclear F-actin formations can affect the RNAPII transcription and condensation. Overall, our findings shed light on the role of PIP2 in RNAPII transcription through identifying the F-actin binding protein MPRIP as a transcription regulator and a determinant of the condensation of RNAPII.
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Wu, Yuhong, Jiang Zhu, Xiaolan Huang, and Zhihua Du. "Crystal structure of a dimerization domain of human Caprin-1: insights into the assembly of an evolutionarily conserved ribonucleoprotein complex consisting of Caprin-1, FMRP and G3BP1." Acta Crystallographica Section D Structural Biology 72, no. 6 (May 25, 2016): 718–27. http://dx.doi.org/10.1107/s2059798316004903.
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Caprin-1 plays roles in many important biological processes, including cellular proliferation, innate immune response, stress response and synaptic plasticity. Caprin-1 has been implicated in several human diseases, including osteosarcoma, breast cancer, viral infection, hearing loss and neurodegenerative disorders. The functions of Caprin-1 depend on its molecular-interaction network. Direct interactions have been established between Caprin-1 and the fragile X mental retardation protein (FMRP), Ras GAP-activating protein-binding protein 1 (G3BP1) and theJapanese encephalitis virus(JEV) core protein. Here, crystal structures of a fragment (residues 132–251) of Caprin-1, which adopts a novel all-α-helical fold and mediates homodimerization through a substantial interface, are reported. Homodimerization creates a large and highly negatively charged concave surface suggestive of a protein-binding groove. The FMRP-interacting sequence motif forms an integral α-helix in the dimeric Caprin-1 structure in such a way that the binding of FMRP would not disrupt the homodimerization of Caprin-1. Based on insights from the structures and existing biochemical data, the existence of an evolutionarily conserved ribonucleoprotein (RNP) complex consisting of Caprin-1, FMRP and G3BP1 is proposed. The JEV core protein may bind Caprin-1 at the negatively charged putative protein-binding groove and an adjacent E-rich sequence to hijack the RNP complex.
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Wang, Qiang, Yi Xie, Quan-Sheng Du, Xiao-Jun Wu, Xu Feng, Lin Mei, Jay M. McDonald, and Wen-Cheng Xiong. "Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin." Journal of Cell Biology 160, no. 4 (February 10, 2003): 565–75. http://dx.doi.org/10.1083/jcb.200207036.
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Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.
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Nie, Zuqin, Yutong Xue, Dafeng Yang, Sharleen Zhou, Bonnie J. Deroo, Trevor K. Archer, and Weidong Wang. "A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex." Molecular and Cellular Biology 20, no. 23 (December 1, 2000): 8879–88. http://dx.doi.org/10.1128/mcb.20.23.8879-8888.2000.
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ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.
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Weighardt, F., F. Cobianchi, L. Cartegni, I. Chiodi, A. Villa, S. Riva, and G. Biamonti. "A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock." Journal of Cell Science 112, no. 10 (May 15, 1999): 1465–76. http://dx.doi.org/10.1242/jcs.112.10.1465.
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A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.
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Kumar, Maneesh G., Neil M. Patel, Adam M. Nicholson, Amanda L. Kalen, Ehab H. Sarsour, and Prabhat C. Goswami. "Reactive oxygen species mediate microRNA-302 regulation of AT-rich interacting domain 4a and C-C motif ligand 5 expression during transitions between quiescence and proliferation." Free Radical Biology and Medicine 53, no. 4 (August 2012): 974–82. http://dx.doi.org/10.1016/j.freeradbiomed.2012.06.019.
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Ling, Pin, Zhengbin Yao, Christian F. Meyer, Xuhong Sunny Wang, Wolf Oehrl, Stephan M. Feller, and Tse-Hua Tan. "Interaction of Hematopoietic Progenitor Kinase 1 with Adapter Proteins Crk and CrkL Leads to Synergistic Activation of c-Jun N-Terminal Kinase." Molecular and Cellular Biology 19, no. 2 (February 1, 1999): 1359–68. http://dx.doi.org/10.1128/mcb.19.2.1359.
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ABSTRACT Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.
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Nikko, Elina, and Bruno André. "Split-Ubiquitin Two-Hybrid Assay To Analyze Protein-Protein Interactions at the Endosome: Application to Saccharomyces cerevisiae Bro1 Interacting with ESCRT Complexes, the Doa4 Ubiquitin Hydrolase, and the Rsp5 Ubiquitin Ligase." Eukaryotic Cell 6, no. 8 (May 18, 2007): 1266–77. http://dx.doi.org/10.1128/ec.00024-07.
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ABSTRACT Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.
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Masters, Thomas A., and Folma Buss. "Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI." Proceedings of the National Academy of Sciences 114, no. 7 (January 31, 2017): 1595–600. http://dx.doi.org/10.1073/pnas.1616941114.
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Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end–directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end–directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.
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Heusipp, Gerhard, Katrin Spekker, Sabine Brast, Stefan Fälker, and M. Alexander Schmidt. "YopM of Yersinia enterocolitica specifically interacts with α1-antitrypsin without affecting the anti-protease activity." Microbiology 152, no. 5 (May 1, 2006): 1327–35. http://dx.doi.org/10.1099/mic.0.28697-0.
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It was previously shown that α1-antitrypsin (AAT) interacts with the type III secreted (T3S) EspB and EspD proteins of enteropathogenic Escherichia coli (EPEC), resulting in reduced functionality of the proteins. To determine if AAT is also able to interact with T3S proteins of other pathogens, the binding of AAT to Yop proteins of Yersinia enterocolitica was analysed. AAT did not interact with YopB or YopD, which have functions in type III translocation similar to EspB and EspD in EPEC, but specifically interacts with YopM, a member of the leucine-rich repeat (LRR) family of proteins, in overlay and pull-down assays. To determine regions of YopM involved in AAT binding, various N- and C-terminally truncated versions of YopM were recombinantly expressed, and their ability to interact with AAT analysed. All versions tested were able to bind AAT, indicating that at least eight LRR of YopM are sufficient for AAT interaction. The main physiological role of AAT is to inhibit neutrophil elastase; however, elastase was efficiently inhibited by AAT in the presence and absence of YopM, indicating that YopM does not interfere with the anti-protease inhibition activity of AAT, and that the domain of AAT interacting with YopM is not identical to AAT's protease interaction domain. Furthermore, it was shown that elastase efficiently degrades YopM and other Yop proteins. The data suggest that AAT has additional functions in the host response against bacterial infections that are not related to its anti-protease activity.
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Huber, Sandra, Tulin Karagenc, Dominic Ritler, Sven Rottenberg, and Kerry Woods. "Identification and characterisation of aTheileria annulataproline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface." Cellular Microbiology 20, no. 7 (April 3, 2018): e12838. http://dx.doi.org/10.1111/cmi.12838.
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Coughlin, Michael W., Joshua S. Bloom, Guy Nir, Sarah Antier, Theophile Jegou du Laz, Stéfan van der Walt, Arien Crellin-Quick, et al. "A Data Science Platform to Enable Time-domain Astronomy." Astrophysical Journal Supplement Series 267, no. 2 (July 31, 2023): 31. http://dx.doi.org/10.3847/1538-4365/acdee1.
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Abstract SkyPortal is an open-source software package designed to discover interesting transients efficiently, manage follow-up, perform characterization, and visualize the results. By enabling fast access to archival and catalog data, crossmatching heterogeneous data streams, and the triggering and monitoring of on-demand observations for further characterization, a SkyPortal-based platform has been operating at scale for >2 yr for the Zwicky Transient Facility Phase II community, with hundreds of users, containing tens of millions of time-domain sources, interacting with dozens of telescopes, and enabling community reporting. While SkyPortal emphasizes rich user experiences across common front-end workflows, recognizing that scientific inquiry is increasingly performed programmatically, SkyPortal also surfaces an extensive and well-documented application programming interface system. From back-end and front-end software to data science analysis tools and visualization frameworks, the SkyPortal design emphasizes the reuse and leveraging of best-in-class approaches, with a strong extensibility ethos. For instance, SkyPortal now leverages ChatGPT large language models to generate and surface source-level human-readable summaries automatically. With the imminent restart of the next generation of gravitational-wave detectors, SkyPortal now also includes dedicated multimessenger features addressing the requirements of rapid multimessenger follow-up: multitelescope management, team/group organizing interfaces, and crossmatching of multimessenger data streams with time-domain optical surveys, with interfaces sufficiently intuitive for newcomers to the field. This paper focuses on the detailed implementations, capabilities, and early science results that establish SkyPortal as a community software package ready to take on the data science challenges and opportunities presented by this next chapter in the multimessenger era.
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Lavillette, Dimitri, Marielle Maurice, Catherine Roche, Stephen J. Russell, Marc Sitbon, and François-Loïc Cosset. "A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes." Journal of Virology 72, no. 12 (December 1, 1998): 9955–65. http://dx.doi.org/10.1128/jvi.72.12.9955-9965.1998.
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ABSTRACT The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.
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Nile, Arti, Jisoo Shin, Juhyun Shin, Gyun Seok Park, Suhyun Lee, Ji-Ho Lee, Kyung-Woo Lee, et al. "Cinnamaldehyde-Rich Cinnamon Extract Induces Cell Death in Colon Cancer Cell Lines HCT 116 and HT-29." International Journal of Molecular Sciences 24, no. 9 (May 3, 2023): 8191. http://dx.doi.org/10.3390/ijms24098191.
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Cinnamon is a natural spice with a wide range of pharmacological functions, including anti-microbial, antioxidant, and anti-tumor activities. The aim of this study is to investigate the effects of cinnamaldehyde-rich cinnamon extract (CRCE) on the colorectal cancer cell lines HCT 116 and HT-29. The gas chromatography mass spectrometry analysis of a lipophilic extract of cinnamon revealed the dominance of trans-cinnamaldehyde. Cells treated with CRCE (10–60 µg/mL) showed significantly decreased cell viability in a time- and dose-dependent manner. We also observed that cell proliferation and migration capacity were inhibited in CRCE-treated cells. In addition, a remarkable increase in the number of sub-G1-phase cells was observed with arrest at the G2 phase by CRCE treatment. CRCE also induced mitochondrial stress, and finally, CRCE treatment resulted in activation of apoptotic proteins Caspase-3, -9, and PARP and decreased levels of mu-2-related death-inducing gene protein expression with BH3-interacting domain death agonist (BID) activation.
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Xiong, Wei, Jie Yang, Mingzhen Wang, Hailong Wang, Zhipeng Rao, Cheng Zhong, Xiu Xin, et al. "Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation." Journal of Virology 89, no. 14 (May 13, 2015): 7385–400. http://dx.doi.org/10.1128/jvi.00567-15.
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ABSTRACTHepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin β coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin β and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin β significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin β short hairpin RNA-resistant mutant. We also found that vinexin β modulated the hyperphosphorylation of NS5A in a casein kinase 1α-dependent on manner. Taken together, our findings suggest that vinexin β modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin β in the viral life cycle.IMPORTANCEHepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin β modulates NS5A hyperphosphorylation through the NS5A-vinexin β interaction. Hyperphosphorylated NS5A induced by vinexin β is casein kinase 1α dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight onFlaviviridaeNS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.
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Brunet, Geneviève M., Edith Gagnon, Charles F. Simard, Nikolas D. Daigle, Luc Caron, Micheline Noël, Marie-Hélène Lefoll, Marc J. Bergeron, and Paul Isenring. "Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants." Journal of General Physiology 126, no. 4 (September 12, 2005): 325–37. http://dx.doi.org/10.1085/jgp.200509334.
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The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.
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Asano, Haruhiko, Xi Susan Li, and George Stamatoyannopoulos. "FKLF-2: a novel Krüppel-like transcriptional factor that activates globin and other erythroid lineage genes." Blood 95, no. 11 (June 1, 2000): 3578–84. http://dx.doi.org/10.1182/blood.v95.11.3578.011k48_3578_3584.
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FKLF-2, a novel Krüppel-type zinc finger protein, was cloned from murine yolk sac. The deduced polypeptide sequence of 289 amino acids has 3 contiguous zinc fingers at the near carboxyl-terminal end, an amino-terminal domain characterized by its high content of alanine and proline residues and a carboxyl-terminal domain rich in serine residues. By Northern blot hybridization, the human homologue of FKLF-2 is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in established cell lines with an erythroid phenotype, but it is inconsistently expressed in cell lines with myeloid or lymphoid phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is up-regulated after induction of mouse erythroleukemia cells. In luciferase assays, FKLF-2 activates predominantly the γ, and to a lesser degree, the ɛ and β globin gene promoters. The activation of γ gene promoter does not depend on the presence of an HS2 enhancer. FKLF-2 activates the γ promoter predominantly by interacting with the γ CACCC box, and to a lesser degree through interaction with the TATA box or its surrounding DNA sequences. FKLF-2 also activated all the other erythroid specific promoters we tested (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase). These results suggest that in addition to globin, FKLF-2 may be involved in activation of transcription of a wide range of genes in the cells of the erythroid lineage.
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Filipowska, Joanna K., Nagesha G. Kondegowda, and Rupangi C. Vasavada. "LGR4 and Its Extracellular Domain as Novel Regulators of ß-Cell Survival and Proliferation." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A321—A322. http://dx.doi.org/10.1210/jendso/bvab048.656.
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Abstract Our lab has shown that RANK (Receptor activator of the NF-κB) by interacting with its ligand, RANKL, inhibits ß-cell proliferation and survival; which can be reversed by Osteoprotegerin (OPG). Recently, the G protein-coupled receptor LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4), which binds R-spondin (RSPO), was identified as a novel receptor for RANKL in osteoclast precursor cells. Thus, RANKL can bind two distinct receptors, RANK and LGR4 in osteoclasts, leading to opposite effects on osteoclastogenesis. LGR4 is expressed in rodent and human ß-cells, but the role of this receptor in ß-cells remains unknown. We postulated that LGR4 through its interaction with RANKL is involved in regulating ß-cell survival and proliferation. Our data indicate expression of specific LGR4 family members, Lgr4, Rank, Rankl, is modulated by stressors, such as cytokines, ER stress, diabetes and aging, in INS1 cells, rodent and human islets. Knocking down Lgr4 in INS1 cells or rodent islets has no significant effect on ß-cell proliferation but is detrimental for ß-cell survival in basal and cytokine-stimulated conditions. We also propose that the soluble extracellular domain of LGR4 (LGR4-ECD), which binds to its ligands (RSPO/RANKL), holds therapeutic potential like OPG, by inhibiting the interaction between RANKL/RANK. At 200ng/ml LGR4-ECD significantly enhances young adult (8-12-week-old) and aged (1.y.o.) rodent ß-cell proliferation, as well as human ß-cell proliferation, in islets from not only control subjects (45±17 y.o.), but also with Type 2 diabetes (48±7 y.o.). Additionally, LGR4-ECD significantly promotes mouse and human ß-cell survival against cytokine-induced cell death. Future studies will determine the physiological role of LGR4 and the therapeutic potential of LGR4-ECD on the beta cell in vivo in basal conditions and in the setting of diabetes. Acknowledgements: Funding: JDRF postdoctoral fellowship # 3-PDF-2020-936-A-N to JF; Human Islets: IIDP
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Davis, Beckley K., Taylor Baker, Alma Rechnitzer, Matthew Hamby, Zachary Troiani, Sarah Stenske, Samuel Davis, and Anna Bauer. "NLRC3 Localizes to the Endoplasmic Reticulum via Interactions with a Novel ER-Resident Protein." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 68.18. http://dx.doi.org/10.4049/jimmunol.204.supp.68.18.
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Abstract Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I interferon. One of several upstream receptor cGAS (cyclic-GMP-AMP synthase) binds to cytosolic DNA and generates di-cyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to Stimulator of Interferon Genes (STING). STING recruits TANK binding kinase 1 (TBK1) which acts as a critical node that allows for efficient activation of interferon regulatory factors (IRFs) to drive the anti-viral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine rich repeat containing protein (NLR) that negatively regulates the type I interferon pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I interferons. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two hybrid screening. Here we show that a novel ER-resident protein associates with NLRC3 in human cells. This interaction occurs at the ER. This data provides a mechanism by which NLRC3 localizes to the ER to affect STING signaling in response to cytosolic nucleotides.
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Garland, Kathleen, Owen J. T. McCarty, and Cristina Puy. "Proteolytic Inactivation of ADAMTS13 By Activated Factor XI." Blood 128, no. 22 (December 2, 2016): 4955. http://dx.doi.org/10.1182/blood.v128.22.4955.4955.
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Abstract Background: ADAMTS13, a plasma metalloprotease, is secreted into blood as an active enzyme that cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular flow. Plasma ADAMTS13 has a molecular weight of 200 kDa, consisting of a metalloprotease (MET) domain, a disintegrin-like domain, a first thrombospondin type-1 repeat (TSP1) domain, a Cys-rich domain, and a spacer domain. Moreover, the C-terminal domain of ADAMTS13 contains an additional seven TSP1 repeats and two CUB domains. ADAMTS13 has been shown to adopt a natural folded conformation, allowing its CUB domains to interact with its spacer domain. This more closed conformation prohibits the functional exosite on the spacer domain from interacting with its proteolytic site on the A2 domain of VWF. In plasma, globular ADAMTS13 will associate with VWF via necessary binding of the CUB domains to the VWF D4CK fragment. Under shear stress or flow conditions, bound ADAMTS13 will unfold leading to exposure of the spacer domain exosite and ultimately increased ADAMTS13 proteolysis VWF. Without the CUB domains, ADAMTS13 does not proteolyze VWF under flow conditions. To date, it is still uncertain how ADAMTS13 activity is regulated, and what impact this has on the inactivation of VWF. The serine proteases thrombin, activated FX (FXa), and plasmin have been shown to cleave and inactivate ADAMTS13. Based on the fact that congenital factor XI deficiencies are associated with bleeding disorders and that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that the serine protease activated FXI (FXIa) inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine whether FXIa is able to cleave and inactivate ADAMTS13. Methods and results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0-3 hours) at 37oC before being separated by SDS-PAGE and analyzed by Coomassie blue staining, resulting in the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of lower molecular weight bands under reducing conditions. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13 by FXIa. Samples were analyzed by western blot to determine the cleavage site using an anti-ADAMTS13 antibody, which specifically binds the two CUB domains, and an anti-ADAMTS13 antibody which specifically binds the MET domain. ADAMTS13 has been shown to be cleaved by serine proteases, such as plasmin, thrombin and FXa. We incubated ADAMTS13 (200nM) with equivalent concentrations of plasmin, thrombin, FXa, FXIa, FXIIa or Kallikrein at 37oC over a time interval of 0-3 hours. The addition of Ca++(5 mM) was necessary for proteolytic activity of thrombin and FXa. We observed that the ability of FXIa to cleave ADAMTS13 was found to be similar to the ability of thrombin to cleave ADAMTS13. Neither FXa, kallikrein, nor FXIIa appeared to cleave ADAMTS13. The antibody against the MET domain detected a single broad band at approximately 150 kDa. When the samples were analyzed with the antibody specific for the two CUB domains, a single broad band at approximately 50 kDa was detected, suggesting that the proteolysis of ADAMTS13 by FXIa preferentially occurs near the start of the first CUB domain. Interestingly, it has been previously reported that the CUB domains are necessary for VWF strand cleavage under flow conditions. It has been shown that the cleavage of a fluorescence-quenching substrate, FRETS-VWF73, by ADAMTS13 was enhanced after CUB1-2 domain removal. We observed that after the incubation of ADAMTS13 (30nM) with FXIa (30nM) at 37oC for 3 hours, the activity of ADAMTS13 was increased. Analysis of the samples by western blot using an anti-ADAMTS13 MET antibody confirmed the generation of the 150 kDa fragment. Conclusion: Our study suggests a novel molecular link between the regulation of VWF activity and FXI through inactivation of ADAMTS13. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also through limiting ADAMTS13-mediated VWF inactivation. Our future studies are focused on determining the physiological role of the proteolytic removal of the CUB domains of ADAMTS13 by FXIa under flow conditions by measuring platelet aggregation. Disclosures No relevant conflicts of interest to declare.