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1
Walz, J., A. J. Koster, T. Tamura, and W. Baumeister. "Macromolecular Assemblies Designed for Controlled Proteolysis." Microscopy and Microanalysis 4, S2 (July 1998): 980–81. http://dx.doi.org/10.1017/s1431927600025022.
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Since cellular structures are rebuilt continually, protein degradation is essential for the maintenance of homeostasis. Misfolded proteins ensuing from genetic defects or environmental stress, are prone to aggregation; chaperones and proteases cooperate in minimizing such unproductive interactions. Last, but not least, protein degradation serves to terminate the lifespan of many regulatory proteins at distinct times and thus features as a key regulatory element itself. Proteins destined for degradation must be recognized and selected within the crowded environment of the cell. The stratagem of self-compartmentalization is key to controlling cellular proteolysis (1).In recent years, a number of multisubunit proteolytic complexes have been described which possess large internal cavities or nano-compartments. This allows them to confine the proteolytic action to their interior; access to these inner compartments is usually restricted to the unfolded proteins. This, in turn, makes it necessary for these proteases to interact - either in a transient or in a permanent manner
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Koppen, Mirko, Metodi D. Metodiev, Giorgio Casari, Elena I. Rugarli, and Thomas Langer. "Variable and Tissue-Specific Subunit Composition of Mitochondrial m-AAA Protease Complexes Linked to Hereditary Spastic Paraplegia." Molecular and Cellular Biology 27, no. 2 (November 13, 2006): 758–67. http://dx.doi.org/10.1128/mcb.01470-06.
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ABSTRACT The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.
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Aweida, Dina, and Shenhav Cohen. "Breakdown of Filamentous Myofibrils by the UPS–Step by Step." Biomolecules 11, no. 1 (January 15, 2021): 110. http://dx.doi.org/10.3390/biom11010110.
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Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.
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Yang, Dongsik, Hongjian He, and Bing Xu. "Enzyme-instructed morphological transition of the supramolecular assemblies of branched peptides." Beilstein Journal of Organic Chemistry 16 (November 4, 2020): 2709–18. http://dx.doi.org/10.3762/bjoc.16.221.
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Here, we report the use of an enzymatic reaction to cleave the branch off branched peptides for inducing the morphological transition of the assemblies of the peptides. The attachment of DEDDDLLI sequences to the ε-amine of the lysine residue of a tetrapeptide produces branched peptides that form micelles. Upon the proteolytic cleavage of the branch, catalyzed by proteinase K, the micelles turn into nanofibers. We also found that the acetylation of the N-terminal of the branch increased the stability of the branched peptides. Moreover, these branched peptides facilitate the delivery of the proteins into cells. This work contributes insights for the development of peptide supramolecular assemblies via enzymatic noncovalent synthesis in cellular environment.
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Oláh, Judit, Attila Lehotzky, Tibor Szénási, and Judit Ovádi. "Anti-Aggregative Effect of the Antioxidant DJ-1 on the TPPP/p25-Derived Pathological Associations of Alpha-Synuclein." Cells 10, no. 11 (October 27, 2021): 2909. http://dx.doi.org/10.3390/cells10112909.
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DJ-1, a multi-functional protein with antioxidant properties, protects dopaminergic neurons against Parkinson’s disease (PD). The oligomerization/assembly of alpha-synuclein (SYN), promoted by Tubulin Polymerization Promoting Protein (TPPP/p25), is fatal in the early stage of PD. The pathological assembly of SYN with TPPP/p25 inhibits their proteolytic degradation. In this work, we identified DJ-1 as a new interactive partner of TPPP/p25, and revealed its influence on the association of TPPP/p25 with SYN. DJ-1 did not affect the TPPP/p25-derived tubulin polymerization; however, it did impede the toxic assembly of TPPP/p25 with SYN. The interaction of DJ-1 with TPPP/p25 was visualized in living human cells by fluorescence confocal microscopy coupled with Bifunctional Fluorescence Complementation (BiFC). While the transfected DJ-1 displayed homogeneous intracellular distribution, the TPPP/p25-DJ-1 complex was aligned along the microtubule network. The anti-aggregative effect of DJ-1 on the pathological TPPP/p25-SYN assemblies was established by the decrease in the intensity of their intracellular fluorescence (BiFC signal) and the increase in the proteolytic degradation of SYN complexed with TPPP/p25 due to the DJ-1-derived disassembly of SYN with TPPP/p25. These data obtained with HeLa and SH-SY5Y cells revealed the protective effect of DJ-1 against toxic SYN assemblies, which assigns a new function to the antioxidant sensor DJ-1.
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Uskoković, V., M.-K. Kim, W. Li, and S. Habelitz. "Enzymatic processing of amelogenin during continuous crystallization of apatite." Journal of Materials Research 23, no. 12 (December 2008): 3184–95. http://dx.doi.org/10.1557/jmr.2008.0387.
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Dental enamel forms through a protein-controlled mineralization and enzymatic degradation process with a nanoscale precision that new engineering technologies may be able to mimic. Recombinant full-length human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were used in a pH-stat titration system that enabled a continuous supply of calcium and phosphate ions over several days, mimicking the initial stages of matrix processing and crystallization in enamel in vitro. Effects on the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/mL rH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. A transition from nanospheres to fibrous amelogenin assemblies was facilitated under conditions that involved interaction between rH174 and its proteolytic cleavage products. Despite continuous titration, the levels of calcium exhibited a consistent trend of decreasing, thereby indicating a possible role in protein self-assembly. This study suggests that mimicking enamel formation in vitro requires the synergy between the aspects of matrix self-assembly, proteolysis, and crystallization.
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Yang, Jin-Kyoung, Seon-Yeong Kwak, Su-Ji Jeon, Eunjin Lee, Jong-Min Ju, Hye-In Kim, Yoon-Sik Lee, and Jong-Ho Kim. "Proteolytic disassembly of peptide-mediated graphene oxide assemblies for turn-on fluorescence sensing of proteases." Nanoscale 8, no. 24 (2016): 12272–81. http://dx.doi.org/10.1039/c6nr02815b.
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Erk, Inge, Jean-Claude Huet, Mariela Duarte, Stéphane Duquerroy, Felix Rey, Jean Cohen, and Jean Lepault. "A Zinc Ion Controls Assembly and Stability of the Major Capsid Protein of Rotavirus." Journal of Virology 77, no. 6 (March 15, 2003): 3595–601. http://dx.doi.org/10.1128/jvi.77.6.3595-3601.2003.
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ABSTRACT The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH2-terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.
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Harkness, Robert W., Yuki Toyama, Zev A. Ripstein, Huaying Zhao, Alexander I. M. Sever, Qing Luan, Jacob P. Brady, Patricia L. Clark, Peter Schuck, and Lewis E. Kay. "Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP." Proceedings of the National Academy of Sciences 118, no. 32 (August 6, 2021): e2109732118. http://dx.doi.org/10.1073/pnas.2109732118.
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DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a “resting” hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP’s substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.
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Tamara, Sem, Vojtech Franc, and Albert J. R. Heck. "A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin." Proceedings of the National Academy of Sciences 117, no. 27 (June 19, 2020): 15554–64. http://dx.doi.org/10.1073/pnas.2002483117.
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The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1β units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(β)noligomers (n> 1) while Hp 2-2 occurs in cyclic (α2)n(β)noligomers (n> 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp’s antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp’s capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor’s genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.
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Osman, Christof, Claudia Wilmes, Takashi Tatsuta, and Thomas Langer. "Prohibitins Interact Genetically with Atp23, a Novel Processing Peptidase and Chaperone for the F1FO-ATP Synthase." Molecular Biology of the Cell 18, no. 2 (February 2007): 627–35. http://dx.doi.org/10.1091/mbc.e06-09-0839.
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The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F1FO-ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial-encoded FO-subunit Atp6 after its insertion into the inner membrane. On the other hand and independent of its proteolytic activity, Atp23 promotes the association of mature Atp6 with Atp9 oligomers. This assembly step is thus under the control of two substrate-specific chaperones, Atp10 and Atp23, which act on opposite sides of the inner membrane. Strikingly, both ATP10 and ATP23 were found to genetically interact with prohibitins, which build up large, ring-like assemblies with a proposed scaffolding function in the inner membrane. Our results therefore characterize not only a novel processing peptidase with chaperone activity in the mitochondrial intermembrane space but also link the function of prohibitins to the F1FO-ATP synthase complex.
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Ehses, Sarah, Ines Raschke, Giuseppe Mancuso, Andrea Bernacchia, Stefan Geimer, Daniel Tondera, Jean-Claude Martinou, Benedikt Westermann, Elena I. Rugarli, and Thomas Langer. "Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1." Journal of Cell Biology 187, no. 7 (December 28, 2009): 1023–36. http://dx.doi.org/10.1083/jcb.200906084.
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Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)–dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.
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Gres, Anna, Karen Kirby, Atsuko Hachiya, Eleftherios Michailidis, Owen Pornillos, Wataru Sugiura, KyeongEun Lee, Vineet KewalRamani, John Tanner, and Stefan Sarafianos. "Native Hexameric Full-Length HIV-1 Capsid: Crystal Structure and Drug Targeting." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C696. http://dx.doi.org/10.1107/s2053273314093036.
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The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.
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Baier, Kerstin, Heike Lehmann, Dirk Paul Stephan, and Wolfgang Lockau. "NblA is essential for phycobilisome degradation in Anabaena sp. strain PCC 7120 but not for development of functional heterocysts." Microbiology 150, no. 8 (August 1, 2004): 2739–49. http://dx.doi.org/10.1099/mic.0.27153-0.
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Phycobilisomes (PBS) are the major light-harvesting complexes of cyanobacteria. These usually blue-coloured multiprotein assemblies are rapidly degraded when the organisms are starved for combined nitrogen. This proteolytic process causes a colour change of the cyanobacterial cells from blue-green to yellow-green (‘bleaching’). As is well documented for the unicellular, non-diazotrophic cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, a gene termed nblA plays a key role in PBS degradation. Filamentous, diazotrophic cyanobacteria like Anabaena adapt to nitrogen deprivation by differentiation of N2-fixing heterocysts. However, during the first hours after nitrogen deprivation all cells degrade their PBS. When heterocysts mature and nitrogenase becomes active, vegetative cells resynthesize their light-harvesting complexes while in heterocysts the phycobiliprotein content remains very low. Expression and function of nblA in Anabaena sp. PCC 7120 was investigated. This strain has two nblA homologous genes, one on the chromosome (nblA) and one on plasmid delta (nblA-p). Northern blot analysis indicated that only the chromosomal nblA gene is up-regulated upon nitrogen starvation. Mutants with interrupted nblA and nblA-p genes, respectively, grew on N2 and developed functional heterocysts. Mutant ΔnblA-p behaved like the wild-type. However, mutant ΔnblA was unable to degrade its PBS, which was most obvious in non-bleaching heterocysts. The results show that NblA, encoded by the chromosomal nblA gene, is required for PBS degradation in Anabaena but is not essential for heterocyst differentiation.
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Yewdell, J., C. Lapham, I. Bacik, T. Spies, and J. Bennink. "MHC-encoded proteasome subunits LMP2 and LMP7 are not required for efficient antigen presentation." Journal of Immunology 152, no. 3 (February 1, 1994): 1163–70. http://dx.doi.org/10.4049/jimmunol.152.3.1163.
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Abstract LMP2 and LMP7 are proteins encoded by MHC genes that are tightly linked to the genes encoding TAP, the transporter that conveys peptides from the cytosol to the endoplasmic reticulum for assembly with MHC class I molecules. LMP2 and LMP7 are subunits of a subset of proteasomes, large molecular assemblies with multi-proteolytic activities believed to degrade damaged and unwanted cellular proteins. Like TAP and class I molecules themselves, expression of LMP genes is enhanced after exposure of cells to IFN-gamma. These findings implicate LMP2 and LMP7 in the cytosolic production of antigenic peptides. Doubts have been cast, however, on the role of LMP2 and LMP7 in Ag processing, because cells lacking these proteins possess class I molecules that contain peptides quantitatively and qualitatively indistinguishable from the peptides bound to class I molecules derived from normal cells. In this paper we show that cells lacking LMP2 and LMP7 present seven TAP-dependent determinants derived from viral proteins. For two determinants, the kinetics of presentation are shown to be similar for LMP-expressing and -nonexpressing cells. We also demonstrate biochemically that peptide is not limiting in the assembly of class I molecules in LMP-nonexpressing cells. These findings provide additional evidence that LMP2 and LMP7 are not required for efficient Ag presentation, and suggest that these proteins have either a more specialized role in the production of class I-associated peptides, or are not involved in the processing of proteins for association with class I molecules.
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Schur, Florian K. M., Robert A. Dick, Wim J. H. Hagen, Volker M. Vogt, and John A. G. Briggs. "The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly." Journal of Virology 89, no. 20 (July 29, 2015): 10294–302. http://dx.doi.org/10.1128/jvi.01502-15.
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ABSTRACTThe polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to studyin vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation.IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.
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Yuan, Dan, Xuewen Du, Junfeng Shi, Ning Zhou, Abdulgader Ahmed Baoum, Khalid Omar Al Footy, Khadija Omar Badahdah, and Bing Xu. "Synthesis and evaluation of the biostability and cell compatibility of novel conjugates of nucleobase, peptidic epitope, and saccharide." Beilstein Journal of Organic Chemistry 11 (August 3, 2015): 1352–59. http://dx.doi.org/10.3762/bjoc.11.145.
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This article reports the synthesis of a new class of conjugates containing a nucleobase, a peptidic epitope, and a saccharide and the evalution of their gelation, biostability, and cell compatibility. We demonstrate a facile synthetic process, based on solid-phase peptide synthesis of nucleopeptides, to connect a saccharide with the nucleopeptides for producing the target conjugates. All the conjugates themselves (1–8) display excellent solubility in water without forming hydrogels. However, a mixture of 5 and 8 self-assembles to form nanofibers and results in a supramolecular hydrogel. The proteolytic stabilities of the conjugates depend on the functional peptidic epitopes. We found that TTPV is proteolytic resistant and LGFNI is susceptible to proteolysis. In addition, all the conjugates are compatible to the mammalian cells tested.
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Wainwright, S. Mark, Ben R. Hopkins, Cláudia C. Mendes, Aashika Sekar, Benjamin Kroeger, Josephine E. E. U. Hellberg, Shih-Jung Fan, et al. "Drosophila Sex Peptide controls the assembly of lipid microcarriers in seminal fluid." Proceedings of the National Academy of Sciences 118, no. 5 (January 25, 2021): e2019622118. http://dx.doi.org/10.1073/pnas.2019622118.
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Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing nonfunctional SP mutant proteins that affect SP’s binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in D. melanogaster.
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Buhimschi, Irina A., Unzila A. Nayeri, Guomao Zhao, Lydia L. Shook, Anna Pensalfini, Edmund F. Funai, Ira M. Bernstein, Charles G. Glabe, and Catalin S. Buhimschi. "Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia." Science Translational Medicine 6, no. 245 (July 16, 2014): 245ra92. http://dx.doi.org/10.1126/scitranslmed.3008808.
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Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state–dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state–dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer’s β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.
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de Sousa, J. P., R. G. Carvalho, L. F. Barbosa-Martins, R. J. S. Torquato, K. C. U. Mugnol, F. D. Nascimento, I. L. S. Tersariol, and R. M. Puppin-Rontani. "The Self-Assembling Peptide P11-4 Prevents Collagen Proteolysis in Dentin." Journal of Dental Research 98, no. 3 (January 5, 2019): 347–54. http://dx.doi.org/10.1177/0022034518817351.
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The major goal in restorative dentistry is to develop a true regenerative approach that fully recovers hydroxyapatite crystals within the caries lesion. Recently, a rationally designed self-assembling peptide P11-4 (Ace-QQRFEWEFEQQ-NH2) has been developed to enhance remineralization on initial caries lesions, yet its applicability on dentin tissues remains unclear. Thus, the present study investigated the interaction of P11-4 with the organic dentin components as well as the effect of P11-4 on the proteolytic activity, mechanical properties of the bonding interface, and nanoleakage evaluation to artificial caries-affected dentin. Surface plasmon resonance and atomic force microscopy indicated that P11-4 binds to collagen type I fibers, increasing their width from 214 ± 4 nm to 308 ± 5 nm ( P < 0.0001). P11-4 also increased the resistance of collagen type I fibers against the proteolytic activity of collagenases. The immediate treatment of artificial caries-affected dentin with P11-4 enhanced the microtensile bonding strength of the bonding interface ( P < 0.0001), reaching values close to sound dentin and decreasing the proteolytic activity at the hybrid layer; however, such effects decreased after 6 mo of water storage ( P < 0.05). In conclusion, P11-4 interacts with collagen type I, increasing the resistance of collagen fibers to proteolysis, and improves stability of the hybrid layer formed by artificial caries-affected dentin.
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Clapp, Aaron R., Ellen R. Goldman, H. Tetsuo Uyeda, Eddie L. Chang, Jessica L. Whitley, and Igor L. Medintz. "Monitoring of Enzymatic Proteolysis Using Self-Assembled Quantum Dot-Protein Substrate Sensors." Journal of Sensors 2008 (2008): 1–10. http://dx.doi.org/10.1155/2008/797436.
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We have previously utilized hybrid semiconductor quantum dot- (QD-) peptide substrates for monitoring of enzymatic proteolysis. In this report, we expand on this sensing strategy to further monitor protein-protease interactions. We utilize QDs self-assembled with multiple copies of dye-labeled proteins as substrates for the sensing of protease activity. Detection of proteolysis is based on changes in the rate of fluorescence resonance energy transfer (FRET) between the QDs and the proximal dye-labeled proteins following protein digestion by added enzyme. Our study focused on two representative proteolytic enzymes: the cysteine protease papain and the serine protease endoproteinase K. Analysis of the enzymatic digestion allowed us to estimate minimal values for the enzymatic activities of each enzyme used. Mechanisms of enzymatic inhibition were also inferred from the FRET data collected in the presence of inhibitors. Potential applications of this technology include drug discovery assays and in vivo cellular monitoring of enzymatic activity.
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DU, Jinping, Barbara H. KNOWLES, Jade LI, and David J. ELLAR. "Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer." Biochemical Journal 338, no. 1 (February 8, 1999): 185–93. http://dx.doi.org/10.1042/bj3380185.
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The interaction of two Bacillus thuringiensiscytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the α-helical N-terminal half and β-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly β-strands was inserted into the membrane, whereas the N-terminal half comprising mainly α-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major β-strands (β-5, β-6 and β-7) in the C-terminal half of the toxin.
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Woodward, Cora L., Sarah N. Cheng, and Grant J. Jensen. "Electron Cryotomography Studies of Maturing HIV-1 Particles Reveal the Assembly Pathway of the Viral Core." Journal of Virology 89, no. 2 (November 12, 2014): 1267–77. http://dx.doi.org/10.1128/jvi.02997-14.
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ABSTRACTTo better characterize the assembly of the HIV-1 core, we have used electron cryotomography (ECT) to image infected cells and the viral particles cryopreserved next to them. We observed progressive stages of virus assembly and egress, including flower-like flat Gag lattice assemblies, hemispherical budding profiles, and virus buds linked to the plasma membrane via a thin membrane neck. The population of budded viral particles contains immature, maturation-intermediate, and mature core morphologies. Structural characteristics of the maturation intermediates suggest that the core assembly pathway involves the formation of a CA sheet that associates with the condensed ribonucleoprotein (RNP) complex. Our analysis also reveals a correlation between RNP localization within the viral particle and the formation of conical cores, suggesting that the RNP helps drive conical core assembly. Our findings support an assembly pathway for the HIV-1 core that begins with a small CA sheet that associates with the RNP to form the core base, followed by polymerization of the CA sheet along one side of the conical core toward the tip, and then closure around the body of the cone.IMPORTANCEDuring HIV-1 assembly and release, the Gag polyprotein is organized into a signature hexagonal lattice, termed the immature lattice. To become infectious, the newly budded virus must disassemble the immature lattice by proteolyzing Gag and then reassemble the key proteolytic product, the structural protein p24 (CA), into a distinct, mature hexagonal lattice during a process termed maturation. The mature HIV-1 virus contains a conical capsid that encloses the condensed viral genome at its wide base. Mutations or small molecules that interfere with viral maturation also disrupt viral infectivity. Little is known about the assembly pathway that results in the conical core and genome encapsidation. Here, we have used electron cryotomography to structurally characterize HIV-1 particles that are actively maturing. Based on the morphologies of core assembly intermediates, we propose that CA forms a sheet-like structure that associates with the condensed viral genome to produce the mature infectious conical core.
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Wileman, T., LP Kane, J. Young, GR Carson, and C. Terhorst. "Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER." Journal of Cell Biology 122, no. 1 (July 1, 1993): 67–78. http://dx.doi.org/10.1083/jcb.122.1.67.
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The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.
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Knorpp, C., C. Szigyarto, and E. Glaser. "Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria." Biochemical Journal 310, no. 2 (September 1, 1995): 527–31. http://dx.doi.org/10.1042/bj3100527.
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We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.
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Qu, Kun, Bärbel Glass, Michal Doležal, Florian K. M. Schur, Brice Murciano, Alan Rein, Michaela Rumlová, Tomáš Ruml, Hans-Georg Kräusslich, and John A. G. Briggs. "Structure and architecture of immature and mature murine leukemia virus capsids." Proceedings of the National Academy of Sciences 115, no. 50 (November 26, 2018): E11751—E11760. http://dx.doi.org/10.1073/pnas.1811580115.
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Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein–protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
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Uskoković, Vuk. "Prospects and Pits on the Path of Biomimetics: The Case of Tooth Enamel." Journal of Biomimetics, Biomaterials and Tissue Engineering 8 (November 2010): 45–78. http://dx.doi.org/10.4028/www.scientific.net/jbbte.8.45.
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This review presents a discourse on challenges in understanding and imitating the process of amelogenesis in vitro on the molecular scale. In light of the analysis of imitation of the growth of dental enamel, it also impends on the prospects and potential drawbacks of the biomimetic approach in general. As the formation of enamel proceeds with the protein matrix guiding the crystal growth, while at the same time conducting its own degradation and removal, it is argued that three aspects of amelogenesis need to be induced in parallel: a) crystal growth; b) protein assembly; c) proteolytic degradation. A particular emphasis is therefore placed on ensuring conditions for proteolysis-coupled protein-guided crystallization to occur. Discussed are structural and functional properties of the protein species involved in amelogenesis, mainly amelogenin and enamelysin, the main protein and the protease of the developing enamel matrix, respectively. A model of enamel growth based on controlled delivery of constituent ions or crystalline or amorphous building blocks by means of amelogenin is proposed. The importance of high viscosity of the enamel matrix and a more intricate role that water may play in such a gelatinous medium are also touched upon. The tendency of amelogenin to self-assemble into fibrous and rod-shaped morphologies is considered as potentially important in explaining the formation of elongated apatite crystals. The idea that a pre-assembling protein matrix serves as a template for the uniaxial growth of apatite crystals in enamel is finally challenged with the one based on co-assembly of the protein and the mineral phases.
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Ferch, Uta, Bernhard Kloo, Andreas Gewies, Vera Pfaender, Michael Düwel, Christian Peschel, Daniel Krappmann, and Jürgen Ruland. "Inhibition of MALT1 Protease Activity Is Selectively Toxic for Activated B CellÃ,–Like Diffuse Large B Cell Lymphoma Cells." Blood 114, no. 22 (November 20, 2009): 1271. http://dx.doi.org/10.1182/blood.v114.22.1271.1271.
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Abstract Abstract 1271 Poster Board I-293 Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell-like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10 and the paracaspase MALT1 for survival. CARD11, BCL10 and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11/BCL10/MALT1 (CBM) complexes couple upstream events to IKK/NF-κB activation. MALT1 possesses in addition a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here we demonstrate pre-assembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL but not in germinal center B cell-like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment. Disclosures No relevant conflicts of interest to declare.
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Na, Mahesheema, Joesph R. Wiencek, Jamila Hirbawi, and Michael Kalafatis. "Cleavage At Arg1018 During Thrombin-Mediated Activation of Coagulation Factor V Is Dispensable." Blood 118, no. 21 (November 18, 2011): 1186. http://dx.doi.org/10.1182/blood.v118.21.1186.1186.
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Abstract Abstract 1186 Blood clotting results in the proteolytic conversion of prothrombin (Pro) to thrombin which in turn will produce the fibrin clot. The proteolytic conversion of Pro to thrombin is catalyzed by the prothrombinase complex which is composed of the enzyme, factor Xa (FXa), the cofactor, factor Va (FVa), assembled on a membrane surface in the presence of divalent metal ions. Factor V (FV), is a multidomain protein (A1-A2-B-A3-C1-C2) with nominal procoagulant activity and is activated by thrombin to FVa through three sequential proteolytic cleavages at Arg709, Arg1018 and Arg1545. To understand the significance of each cleavage for active cofactor formation and prothrombinase function, recombinant factor V molecules were created by site-directed mutagenesis with two out of three cleavage sites mutated simultaneously (to glutamine). We have generated a FV molecule mutated at the Arg709/1018 cleavage sites (FVQQR), a FV molecule mutated at the Arg709/1545 cleavage sites (FVQRQ), a FV molecule mutated at the Arg1018/1545 cleavage sites (FVRQQ), and a FV molecule that is mutated at all three cleavage sites (FVQQQ). These recombinant FV molecules along with wild type FV (FVWT) were transiently expressed in COS7L cells, purified to homogeneity and assessed for their capability to interact with factor Xa following activation by thrombin, and participate in prothrombinase. Pro activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions in the presence of saturating concentrations of FXa were determined. Two-stage clotting assays revealed that while FVQQQ was devoid of clotting activity following incubation with thrombin, FVaQQR, FVaQRQ and FVaRQQ all had impaired clotting activities compared to FVaWT and plasma derived FVa (FVaPLASMA). Kinetic analyses demonstrated that FVaWT had a Kd of 0.25nM for FXa while all other mutant molecules had impaired binding capabilities for FXa. FVaQQQ was severely impaired in its ability to interact with FXa. The kcat value for prothrombinase assembled with FVaQQR was approximately 50% lower than the kcat obtained with prothrombinase assembled with FVaWT, while prothrombinase assembled with FVaQRQ and FVaRQQ had approximately 3-fold reduced catalytic efficiency when compared to the values obtained with prothrombinase assembled with FVaWT. Following incubation with thrombin prothrombinase assembled with FVaQQQ had no cofactor activity. To determine the importance of the cleavage site at Arg1018 for procofactor activation and the function of amino acid region 1000–1008 during proteolysis, several other recombinant molecules were generated. FVRQR is a FV molecule with the mutation Arg1018→Gln, and FVΔ1000-1008 is a mutant FV molecule with region 1000–1008 deleted. We have also generated FVΔ1000-1008/RQR and FVΔ1000-1008/QRQ. Two-stage clotting assays revealed that FVaRQR and FVaΔ1000-1008/RQR have similar clotting activities as FVaWT, whereas FVaQRQ, FVaΔ1000-1008/QRQ are impaired in their clotting activities. Kinetic analyses demonstrated that FVaRQR and FVaΔ1000-1008/RQR have similar affinity for FXa as FVa WT while FVaQRQ and FVaΔ1000-1008/QRQ were impaired in their interaction with factor Xa. The kcat values for prothrombinase assembled with FVaRQR and FVaΔ1000-1008/RQR were similar to the kcat obtained with prothrombinase assembled with FVa WT, while prothrombinase assembled with FVaQRQ and FVaΔ1000-1008/QRQ had 2-fold and 7-fold reduced catalytic efficiency respectively, when compared to the kcat values obtained with prothrombinase assembled with FVaWT. Overall, the data demonstrate that cleavage at both Arg709 and Arg1545 are a prerequisite for expression of optimum cofactor activity. Our data also suggests that cleavage at Arg1018 is redundant for cofactor activity. The role of cleavage at this site by thrombin during procofactor activation remains to be determined. Disclosures: No relevant conflicts of interest to declare.
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Selvaraj, Bethilda Anne, Abdul Razak Mariatulqabtiah, Kok Lian Ho, Chyan Leong Ng, Chean Yeah Yong, and Wen Siang Tan. "Regulation of Proteolytic Activity to Improve the Recovery of Macrobrachium rosenbergii Nodavirus Capsid Protein." International Journal of Molecular Sciences 22, no. 16 (August 13, 2021): 8725. http://dx.doi.org/10.3390/ijms22168725.
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The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
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Serrano, L., F. Wandosell, J. de la Torre, and J. Avila. "Effect of specific proteolytic cleavages on tubulin polymer formation." Biochemical Journal 252, no. 3 (June 15, 1988): 683–91. http://dx.doi.org/10.1042/bj2520683.
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The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.
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Kelly, Jeffery W., and William E. Balch. "Amyloid as a natural product." Journal of Cell Biology 161, no. 3 (May 12, 2003): 461–62. http://dx.doi.org/10.1083/jcb.200304074.
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Amyloid fibrils, such as those found in Alzheimer's and the gelsolin amyloid diseases, result from the misassembly of peptides produced by either normal or aberrant intracellular proteolytic processing. A paper in this issue by Marks and colleagues (Berson et al., 2003) demonstrates that intra-melanosome fibrils are formed through normal biological proteolytic processing of an integral membrane protein. The resulting peptide fragment assembles into fibrils promoting the formation of melanin pigment granules. These results, along with the observation that amyloid fibril formation by bacteria is highly orchestrated, suggest that fibril formation is an evolutionary conserved biological pathway used to generate natural product nanostructures.
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Mitchell, E. J., and A. M. Zimmerman. "Biochemical evidence for the presence of an actin protein in Tetrahymena pyriformis." Journal of Cell Science 73, no. 1 (February 1, 1985): 279–97. http://dx.doi.org/10.1242/jcs.73.1.279.
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A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine. HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8–12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts.
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Saunders, Jared T., and Jean E. Schwarzbauer. "Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing." Molecular Biology of the Cell 30, no. 17 (August 2019): 2218–26. http://dx.doi.org/10.1091/mbc.e19-03-0140.
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The extracellular matrix (ECM) proteins fibronectin (FN) and type I collagen (collagen I) are codistributed in many tissues, and collagens have been shown to depend on an FN matrix for fibrillogenesis. Microscopic analysis of a fibroblast ECM showed colocalization of procollagen I with FN fibrils, and proteolytic cleavage of procollagen to initiate fibril formation was significantly reduced with inhibition of FN matrix assembly. We examined the role of FN matrix in procollagen processing by the C-propeptide proteinase bone morphogenetic protein 1 (BMP-1). We found that BMP-1 binds to a cell-assembled ECM in a dose-dependent manner and that, like procollagen, BMP-1 colocalizes with FN fibrils in the matrix microenvironment. Binding studies with FN fragments identified a binding site in FN’s primary heparin-binding domain. In solution, BMP-1–FN interactions and BMP-1 cleavage of procollagen I were both enhanced by the presence of heparin, suggesting a role for heparin in complex formation during proteolysis. Indeed, addition of heparin enhanced the rate of procollagen cleavage by matrix-bound BMP-1. Our results show that matrix localization of this proteinase facilitates the initiation of collagen assembly and suggest a model in which FN matrix and associated heparan sulfate act as a scaffold to organize enzyme and substrate for procollagen processing.
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Rodell, Christopher B., Ryan J. Wade, Brendan P. Purcell, Neville N. Dusaj, and Jason A. Burdick. "Selective Proteolytic Degradation of Guest–Host Assembled, Injectable Hyaluronic Acid Hydrogels." ACS Biomaterials Science & Engineering 1, no. 4 (March 9, 2015): 277–86. http://dx.doi.org/10.1021/ab5001673.
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Kato, A., K. Yamamoto, S. Miyazaki, SM Jung, M. Moroi, and N. Aoki. "Molecular basis for Glanzmann's thrombasthenia (GT) in a compound heterozygote with glycoprotein IIb gene: a proposal for the classification of GT based on the biosynthetic pathway of glycoprotein IIb-IIIa complex." Blood 79, no. 12 (June 15, 1992): 3212–18. http://dx.doi.org/10.1182/blood.v79.12.3212.3212.
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Abstract The genetic basis for Glanzmann's thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)IIb gene: an opal mutation at the end of exon 17 (CGA----TGA) results in only a trace amount of GPIIb mRNA, and a splicing mutation at the acceptor site of exon 26 (CAG----GAG) causes an in-frame, exon skipping process from exon 25 to 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which includes the proteolytic cleavage site(s) and a unique, proline-rich region at the location corresponding to the carboxyl-terminal of the normal GPIIb alpha-chain. These characteristics are shared by a previously reported defective GPIIb molecule, which is neither assembled with GPIIIa nor transported to the cellular surface. Despite its normal transcription level, expression of the present defective GPIIb molecule was significantly decreased (approximately 6% of the control level). Because the precursor GPIIb molecule is assembled with GPIIIa in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPIIIa subunit, the defective GPIIb molecule may be rapidly degraded by the intrinsic quality control system of the ER due to its inability to form a stable heterodimer complex as a consequence of its misfolded structure. Although we did not confirm that the GPIIIa genes of this individual were normal, GPIIIa may be secondarily decreased (approximately 11% of control), because a large part of it could not be complexed, making it vulnerable to proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPIIb-IIIa complex.
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Kato, A., K. Yamamoto, S. Miyazaki, SM Jung, M. Moroi, and N. Aoki. "Molecular basis for Glanzmann's thrombasthenia (GT) in a compound heterozygote with glycoprotein IIb gene: a proposal for the classification of GT based on the biosynthetic pathway of glycoprotein IIb-IIIa complex." Blood 79, no. 12 (June 15, 1992): 3212–18. http://dx.doi.org/10.1182/blood.v79.12.3212.bloodjournal79123212.
Full textAbstract:
The genetic basis for Glanzmann's thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)IIb gene: an opal mutation at the end of exon 17 (CGA----TGA) results in only a trace amount of GPIIb mRNA, and a splicing mutation at the acceptor site of exon 26 (CAG----GAG) causes an in-frame, exon skipping process from exon 25 to 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which includes the proteolytic cleavage site(s) and a unique, proline-rich region at the location corresponding to the carboxyl-terminal of the normal GPIIb alpha-chain. These characteristics are shared by a previously reported defective GPIIb molecule, which is neither assembled with GPIIIa nor transported to the cellular surface. Despite its normal transcription level, expression of the present defective GPIIb molecule was significantly decreased (approximately 6% of the control level). Because the precursor GPIIb molecule is assembled with GPIIIa in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPIIIa subunit, the defective GPIIb molecule may be rapidly degraded by the intrinsic quality control system of the ER due to its inability to form a stable heterodimer complex as a consequence of its misfolded structure. Although we did not confirm that the GPIIIa genes of this individual were normal, GPIIIa may be secondarily decreased (approximately 11% of control), because a large part of it could not be complexed, making it vulnerable to proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPIIb-IIIa complex.
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Swanekamp, Ria J., Jade J. Welch, and Bradley L. Nilsson. "Proteolytic stability of amphipathic peptide hydrogels composed of self-assembled pleated β-sheet or coassembled rippled β-sheet fibrils." Chem. Commun. 50, no. 70 (2014): 10133–36. http://dx.doi.org/10.1039/c4cc04644g.
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Hydrogel networks composed of rippled β-sheet fibrils of coassembled d- and l-Ac-(FKFE)2-NH2 amphipathic peptides exhibit proteolytic stability and increased rheological strength compared to networks of self-assembled l-Ac-(FKFE)2-NH2 pleated β-sheet fibrils.
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Vilaça, H., G. Pereira, T. G. Castro, B. F. Hermenegildo, J. Shi, T. Q. Faria, N. Micaêlo, et al. "New self-assembled supramolecular hydrogels based on dehydropeptides." Journal of Materials Chemistry B 3, no. 30 (2015): 6355–67. http://dx.doi.org/10.1039/c5tb00501a.
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Mu, C. Jenny, David A. LaVan, Robert S. Langer, and Bruce R. Zetter. "Self-Assembled Gold Nanoparticle Molecular Probes for Detecting Proteolytic Activity In Vivo." ACS Nano 4, no. 3 (February 10, 2010): 1511–20. http://dx.doi.org/10.1021/nn9017334.
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Dodonova, Svetlana O., Simone Prinz, Virginia Bilanchone, Suzanne Sandmeyer, and John A. G. Briggs. "Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses." Proceedings of the National Academy of Sciences 116, no. 20 (April 29, 2019): 10048–57. http://dx.doi.org/10.1073/pnas.1900931116.
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Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation. They encode a Gag polypeptide, the capsid domains of which can oligomerize to form a virus-like particle. The structures of retroviral capsids have been extensively described. They assemble an immature viral particle through oligomerization of full-length Gag. Proteolytic cleavage of Gag results in a mature, infectious particle. In contrast, the absence of structural data on LTR retrotransposon capsids hinders our understanding of their function and evolutionary relationships. Here, we report the capsid morphology and structure of the archetypal Gypsy retrotransposon Ty3. We performed electron tomography (ET) of immature and mature Ty3 particles within cells. We found that, in contrast to retroviruses, these do not change size or shape upon maturation. Cryo-ET and cryo-electron microscopy of purified, immature Ty3 particles revealed an irregular fullerene geometry previously described for mature retrovirus core particles and a tertiary and quaternary arrangement of the capsid (CA) C-terminal domain within the assembled capsid that is conserved with mature HIV-1. These findings provide a structural basis for studying retrotransposon capsids, including those domesticated in higher organisms. They suggest that assembly via a structurally distinct immature capsid is a later retroviral adaptation, while the structure of mature assembled capsids is conserved between LTR retrotransposons and retroviruses.
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Zuber, Guy, Jason McDermott, Sonya Karanjia, Weiyi Zhao, Michael F. Schmid, and Eric Barklis. "Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA." Journal of Virology 74, no. 16 (August 15, 2000): 7431–41. http://dx.doi.org/10.1128/jvi.74.16.7431-7441.2000.
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ABSTRACT Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.
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Yu, I. M., H. A. Holdaway, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen. "Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion." Journal of Virology 83, no. 23 (September 16, 2009): 12101–7. http://dx.doi.org/10.1128/jvi.01637-09.
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ABSTRACT Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.
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Murphy, Brona M., Amanda J. O'Neill, Colin Adrain, R. William G. Watson, and Seamus J. Martin. "The Apoptosome Pathway to Caspase Activation in Primary Human Neutrophils Exhibits Dramatically Reduced Requirements for Cytochrome c." Journal of Experimental Medicine 197, no. 5 (February 24, 2003): 625–32. http://dx.doi.org/10.1084/jem.20021862.
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Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the “apoptosome” as a result of the cell stress–associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell–free extracts readily supported Apaf-1–dependent caspase activation, suggesting that these cells may assemble cytochrome c–independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1–dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1–dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.
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van Asch, Barbara, and Luís Filipe Teixeira da Costa. "Patterns and tempo of PCSK9 pseudogenizations suggest an ancient divergence in mammalian cholesterol homeostasis mechanisms." Genetica 149, no. 1 (January 30, 2021): 1–19. http://dx.doi.org/10.1007/s10709-021-00113-x.
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AbstractProprotein convertase subtilisin/kexin type 9 (PCSK9) plays a central role in cholesterol homeostasis in humans as a major regulator of LDLR levels. PCSK9 is an intriguing protease in that it does not act by proteolysis but by preventing LDLR recirculation from endosomes to the plasma membrane. This, and the inexistence of any other proteolytic substrate but itself could suggest that PCSK9 is an exquisite example of evolutionary fine-tuning. However, the gene has been lost in several mammalian species, and null alleles are present (albeit at low frequencies) in some human populations without apparently deleterious health effects, raising the possibility that the PCSK9 may have become dispensable in the mammalian lineage. To address this issue, we systematically recovered, assembled, corrected, annotated and analysed publicly available PCSK9 sequences for 420 eutherian species to determine the distribution, frequencies, mechanisms and timing of PCSK9 pseudogenization events, as well as the evolutionary pressures underlying the preservation or loss of the gene. We found a dramatic difference in the patterns of PCSK9 retention and loss between Euarchontoglires—where there is strong pressure for gene preservation—and Laurasiatheria, where multiple independent events have led to PCSK9 loss in most species. These results suggest that there is a fundamental difference in the regulation of cholesterol metabolism between Euarchontoglires and Laurasiatheria, which in turn has important implications for the use of Laurasiatheria species (e.g. pigs) as animal models of human cholesterol-related diseases.
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Wickström, Claes, and Ingemar Carlstedt. "N-terminal Cleavage of the Salivary MUC5B Mucin." Journal of Biological Chemistry 276, no. 50 (October 15, 2001): 47116–21. http://dx.doi.org/10.1074/jbc.m106593200.
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Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D′ domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the “soluble” but not in the “insoluble” MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.
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Ryu, Jungki, and Chan Beum Park. "High stability of self-assembled peptide nanowires against thermal, chemical, and proteolytic attacks." Biotechnology and Bioengineering 105, no. 2 (February 1, 2010): 221–30. http://dx.doi.org/10.1002/bit.22544.
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Hall, M. R., and W. Gibson. "Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation." Journal of virology 71, no. 2 (1997): 956–64. http://dx.doi.org/10.1128/jvi.71.2.956-964.1997.
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Genové, Elsa, Nausika Betriu, and Carlos E. Semino. "β-Sheet to Random Coil Transition in Self-Assembling Peptide Scaffolds Promotes Proteolytic Degradation." Biomolecules 12, no. 3 (March 7, 2022): 411. http://dx.doi.org/10.3390/biom12030411.
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One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine.
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Wu, Shenjie, Wenqi Song, Catherine C. L. Wong, and Yigong Shi. "Bax inhibitor 1 is a γ-secretase–independent presenilin-binding protein." Proceedings of the National Academy of Sciences 116, no. 1 (December 17, 2018): 141–47. http://dx.doi.org/10.1073/pnas.1810870116.
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Presenilin is the catalytic subunit of γ-secretase, a four-component intramembrane protease responsible for the generation of β-amyloid (Aβ) peptides. Over 200 Alzheimer’s disease-related mutations have been identified in presenilin 1 (PS1) and PS2. Here, we report that Bax-inhibitor 1 (BI1), an evolutionarily conserved transmembrane protein, stably associates with PS1. BI1 specifically interacts with PS1 in isolation, but not with PS1 in the context of an assembled γ-secretase. The PS1–BI1 complex exhibits no apparent proteolytic activity, as judged by the inability to produce Aβ40 and Aβ42 from the substrate APP-C99. At an equimolar concentration, BI1 has no impact on the proteolytic activity of γ-secretase; at a 200-fold molar excess, BI1 reduces γ-secretase activity nearly by half. Our biochemical study identified BI1 as a PS1-interacting protein, suggesting additional functions of PS1 beyond its involvement in γ-secretase.