Academic literature on the topic 'Ankryin proteins'

Abstract Although proteasome machinery is a conserved cellular component to maintain their normal function, its function in erythrocyte under stress conditions is largely unknown, especially in sickle cell disease (SCD). To determine whether proteasome machinery is altered in SCD erythrocyte, we conducted western blot to detect total ubiquitinated proteins on the erythrocyte membrane in both mice and humans with or without SCD. We found that ubiquitinated proteins were significantly accumulated in SCD mice and humans compared to WT mice and normal controls, indicating that proteasome machinery is halted in SCD. Next, to determine which specific proteins are ubiquitinated and accumulated in SCD, we conducted robust and nonbiased proteomic profiling by immunoprecipitation ubiquitinated proteins followed by proteomics analysis. We found significant accumulation of several categories of ubiquitinated proteins on the erythrocyte membrane in SCD, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), hemoglobin, components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Finally, to determine the effect of halted proteasome machinery in SCD functionally, we conducted in vitro hypoxia induced red blood cell (RBC) sickling assay. We found that inhibition of RBC proteasome machinery by targeting p97 using CB-5083 or targeting proteasome using MG132 increases SCD RBC sickling. Overall, our findings reveal a novel role of halted proteasome machinery in the pathophysiology of SCD and open up new therapies for the disease. Disclosures No relevant conflicts of interest to declare.

Abstract Proteasome machinery is a conserved cellular component to maintain normal protein homeostasis. Hypoxia is well known to control hypoxia inducible factor levels by proteasomal machinery in nucleated cells. However, the specific targets and regulation of proteasomal machinery in non-nucleated mature erythrocytes under hypoxia remain poorly understood. To determine if hypoxia regulates erythrocyte proteasomal machinery, we conducted Western blot to detect total ubiquitinated and K48 specific ubiquitinated proteins on the erythrocyte membrane in both human and mice with or without sickle cell disease (SCD), a hemolytic genetic disease with a high mortality, morbidity and frequently facing hypoxia. We found that ubiquitinated, especially K48 specific ubiquitinated proteins were significantly accumulated in both SCD Berkeley mice and humans compared to WT mice and normal controls, indicating that proteasomal machinery is impaired in SCD. Next, to determine specific ubiquitinated proteins accumulated on the membrane of human sickle erythrocytes (sRBC), we conducted immunoprecipitation of sRBC membrane proteins with total ubiquitin antibody followed by an robust and nonbiased proteomic profiling. We found significant accumulation of several categories of ubiquitinated proteins on human mature sRBC membrane, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), reactive oxygen species (ROS) related enzyme (catalase), components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Mechanistically, we revealed that the impaired proteasomal machinery found in mature sRBC was due to the blockage of trafficking of p97 bound ubiquitinated-proteins from membrane to cytosolic proteasome. As such, inhibition of p97 by CB-5083 or proteasome by MG132 led to further induction of hypoxia-induced ubiquitinated membrane proteins and sickling in cultured human sRBC. Given the fact that sphingosine-1-phosphate (S1P) contributes to sickling by binding with deoxygenated sickle Hb (deoxy-HbS), triggering deoxy-HbS anchoring membrane and releasing glycolytic enzymes, we immediately hypothesized that S1P may be involved in proteasomal machinery by regulating trafficking of p97-bound ubiquitinated proteins from membrane to cytosol in sRBC. To test this intriguing possibility, we generated SCD/Sphk1-/- mouse. Intriguingly, we found that the genetic deletion of SphK1 attenuated impaired proteasomal machinery in sRBC with less accumulation of p97 and ubiquitinated proteins on sRBC membrane, indicating that elevated S1P is detrimental in sRBC by inducing accumulation of p97 and ubiquitinated proteins on the membrane. Moreover, to determine if S1P-regulated p97 trafficking from membrane to the cytosol is unique to sRBC, we exposed wild type and SphK1-/- mice to 8% hypoxia up to 72 hours. In contrast to sRBC, we found that genetic deletion of SphK1 abolished p97 trafficking from membrane to cytosol in normal erythrocytes under hypoxia. Finally, we conducted in vitro proof-of-principle genetic studies to determine if S1P directly involves in translocation of membrane anchored p97 to cytosol using inverted ghost membrane (IGM). We demonstrated that S1P treatment induces deoxy-HbA translocation from cytosol to membrane and in turn restoring p97 release from membrane to the cytosol in IGM isolated from SphK1-/- mice only under hypoxia but not normoxia. Thus, we have provided both human and mouse genetic evidence supporting a working model: in normal individuals under hypoxia, S1P is a key factor regulating the efficient proteasomal machinery by binding deoxy-HbA, promoting deoxy-HbA anchoring membrane and in turn triggering release of p97-ubiqutinated proteins to cytosol for its degradation. With mutation in HbS, S1P promotes deoxy-HbS anchoring the membrane and forms polymers, thus blocks membrane bound p97 release and impairs proteasomal machinery in sRBC. Overall, our findings identified that S1P is a missing key component of proteasome machinery by its differential mechanism regulating p97 trafficking from membrane to cytosol in normal erythrocyte physiology under hypoxia and the pathophysiology of SCD that open up new and differential therapies for the SCD and normal individuals facing hypoxia. Disclosures No relevant conflicts of interest to declare.

Abstract The hematopoietic progenitor cell (HPC) has the potential to differentiate into multiple cell types. Deciphering transcriptional regulatory networks necessary for HPCs to undergo lineage commitment is critical. Toward this goal, we established a serum-free in vitro differentiation system for human HPCs using CD34−positive cells isolated from peripheral blood of normal donors. Cells were cultured in the presence of Epo, SCF and IL-3 for two-weeks, and assessed for total hemoglobin and percent fetal hemoglobin. Adult-derived HPCs express large amounts of Hb A after approximately one week of culture with little expression of Hb F. Transcriptome analysis, using Affymetrix Human Genome U133 Plus 2.0 Arrays on RNA from days 1, 3, 5, 7, 9, 11 and 13 of culture shows more than 1500 differentially-expressed (DE)(> 2-fold up or down) genes, a subset of which were validated independently by real-time RT-PCR. Up-regulated erythroid-specific genes included those for blood group antigens (Kell, Duffy, Rhesus), membrane proteins (ankryin 1, α- and β-spectrin, Band 4.1, Band 4.2, Band 4.9, glycophorin A), receptors (transferrin and erythropoietin receptors), heme biosynthesis enzymes (ALAS, ALAD, UROS, UROD, CPOX, PPOX, FECH), and transcription factors important in globin-gene expression (EKLF, BKLF, NFE2, GATA1) as well as α-, β- and γ-globin mRNAs. Down-regulated genes included those expressed in hematopoietic stem cells (GATA2, CD34) and non-erythroid hematopoietic lineages (MHC class I and II genes, CD33, CD53). The DE genes were clustered into a limited number of expression patterns, suggesting coordinate regulation of genes within each cluster. The DE gene set was examined in silico for co-regulation using transcriptional regulatory network analysis employing PAINT v3.2 software (Vadigepalli R et al., OMICS.2003; 7(3):235–52). GATA binding sites were represented on 149 of the DE gene promoters. Several transcriptional regulatory elements (TRE) were statistically significantly enriched in the DE gene set when compared to the array gene set. These include TRE for AP-1, CREB, GATA-1 and Oct-1. Co-regulation of genes in the different gene clusters will be analyzed by PAINT and findings validated by examination of TRE:transcription factor interactions. These studies should help define timing of transcription factor occupancy in relation to epigenetic changes occurring prior to transcriptome alteration during differentiation of human erythroid cells.

Abstract Abstract 487 Background. Acquired Thrombotic Thrombocytopenic Purpura (TTP) is the result of autoantibodies (Abs) neutralizing and/or accelerating clearance of ADAMTS13. Despite the first-line treatment with daily plasma exchange (PEX), acute TTP is still associated with a mortality rate of ∼20% leaving survivors at high risk of relapse. Availability of molecules for neutralization of pathogenic anti-ADAMTS13 Abs during acute bouts as well as for the targeted elimination of anti-ADAMTS13 specific memory B- and plasma-cells to prevent relapses are thus highly desirable tools to improve patient care and/or treatment of TTP. The primary epitope of pathological relevant, inhibitory anti-ADAMTS13 Abs was identified to lay in the ADAMTS13 spacer domain and to be conformational compromising amino acid residues R568, F592, Y660, Y661 and Y665. We have previously generated 29 specific human monoclonal anti-ADAMTS13 Abs from 2 patients (A and B) splenectomized for frequently relapsing TTP using spleen-derived mononuclear cells (ASH 2011, abstract #194). These anti-ADAMTS13 Abs, characterized by a 5–6 fold increased inhibitory capacity than previously reported as well as eight unique CDR3 motifs in their variable heavy chain genes, four of which are common to both acquired TTP patients, are useful tools to select small anti-idiotypic molecules, mimicking the conformational epitope on ADAMTS13. For this purpose we chose the large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins), characterized by: (1) a high diversity with a library size of 1015–23, allowing to find molecules to any target including conformational epitopes, (2) expressing molecules with a high stability, and (3) being immune tolerant when administered. Methods. Two DARPin libraries, N2C (diversity of 1015) and N3C (1023) coding for two or three randomized ankyrin repeat modules respectively, provided by Molecular Partners AG (Schlieren, Switzerland), were screened against three biotinylated inhibitory anti-ADAMTS13 Abs from patient A belonging to one CDR3 motif (CDR3 motif-1) and pooled in equimolar ratio as targets. Selected DARPins from the fourth round of ribosomal display were cloned, and single clones producing anti-ADAMTS13 idiotypes were purified, tested separately for their specificity towards 29 single, spleen-derived monoclonal anti-ADAMTS13 Abs by ELISA and their DNA sequence analyzed. Results. Seven of 192 (N2C-library) and 2/192 (N3C) single DARPin clones selected against a pool of three inhibitory anti-ADAMTS13 Abs, of a the single CDR3 motif-1, were highly specific for their target. Detailed DNA sequence analysis showed that 4/7 (N2C) and 2/2 (N3C) of the anti-ADAMTS13 specific DARPins were unique whereas 3/7 N2C DARPins consisted of repeats of the unique clones. So far, four (3 N2C and 1 N3C) unique DARPins were purified and their reactivity at equimolar concentration tested towards 200 nM of each of the 29 anti-ADAMTS13 Abs, individually coated on a microtiter plate by ELISA assay. All four DARPins were highly specific for the pool of Abs used for selection as well as other Abs belonging to the same CDR3 motif-1 (n=5). Anti-ADAMTS13 Abs containing the remaining three of the four common (patient A and B) CDR3 motifs, not used for selection, were recognized by 3/4 unique DARPins although to a lesser extent and more anti-ADAMTS13 Abs originating from patient A, were recognized than from patient B, namely 94%, 69% and 56% for CDR3 motif1-3 compared to only 31 %, 54% and 15% from patient B, respectively. Conclusions. Using inhibitory anti-ADAMTS13 of one patient (A) and one CDR3 motif, we were able to select a number of highly specific anti-idiotypic DARPins binding to different monoclonal anti-ADAMTS13 Abs of two genetically and geographically unrelated acquired TTP patients. The fact that several although similar (belonging to the same CDR3 motif) antibodies can be recognized by one anti-idiotypic molecule is promising and hints at a limited number of different anti-idiotypic molecules necessary to neutralize inhibitory anti-ADAMTS13 Abs in the plasma of acquired TTP patients. The analysis of their affinity and the neutralizing potential are ongoing. Disclosures: No relevant conflicts of interest to declare.

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, tissue fibrosis and activation of the innate and adaptive immune system. Clinical features of the disease consist of skin thickening and internal organ involvement. Due to the heterogeneous nature of the disease, there is an unmet need of biomarkers for diagnosis, disease progression and response to treatment.ObjectivesThe aim of this study was to explore new serum proteomic fingerprints of clinically defined forms of SSc.MethodsHighly specific detection of nighty two proteins from a panel related to organ damage was performed, by using the breakthrough technology proximity extension immunoassay (PEA, Olink), in the serum of 72 patients with SSC and 18 age-matched healthy donors (HD). Main disease complications in the SSC cohort, including lung fibrosis, skin fibrosis, renal, vascular, and esophageal involvement were assessed, and prevalence of circulating autoantibodies was tested, along with standard demographic and inflammatory parameters. Unsupervised hierarchical clustering methodologies were applied to identify subgroups of patients based on their proteomic profiles. Gene ontology enrichment was used to interrogate the biological meaning of the distinctive molecular signatures identified.ResultsSixteen circulating proteins related to organ damage were coordinately altered in the serum of SSc patients in relation to HD. Unsupervised clustering analyses differentiated 3 patients clusters presenting different proteomic profiles. Clinically, patients belonging to cluster 1 were characterized by a significant prevalence of multiple organ involvement (84%) in relation to clusters 2 (52%) and 3 (43%), mostly encompassing lung and skin fibrosis and esophageal dysmotility. Immunologically, cluster 1 further displayed the highest percentage of positivity for anti-scl70 antibodies.Nineteen serum proteins, not previously reported in the serum of SSC patients (BANK1, BID, CALR, ERBB2IP, FGR, FOSB, FOXO1, INPPL1, MAEA, MAGED1, MAP4K5, NBN, NCF2, PRKAB1, RASSF2, RCOR1, SMAD1, STXBP3, VASH1) were found deregulated between clusters, with a significant increase in the levels of all of them in cluster 1 compared with clusters 2 and 3. These deregulated proteins were mostly involved in biological processes such as cell proliferation, apoptosis, cell adhesion, migration, and immune response. Among them, two were functionally linked with cutaneous diseases [Calreticulin (CALR) and B-cell scaffold protein with ankryn repeats (BANK1)], two with digestive disorders (Tyrosine-protein kinase Fgr (FGR) and syntaxin-binding protein 3 (STXBP3)] and three with lung disfunction [protein FosB (FOSB), mothers against decapentaplegic homolog 1 (SMAD1) and forkhead box protein O1 (FOXO1)]. Interestingly, levels of some overexpressed proteins in C1 [BH3-interacting domain death agonist (BID), phosphatidylinositol 3,4,5-triphosphate 5-phosphatase 2 (INPPL1), Erbin (ERBB2IP), BANK1 and FOSB] were further related to the positivity for anti-scl70, the specific SSC-autoantibody known to be mostly associated to a bad prognosis and multiple organ involvement in SSC patients.Conclusion:1) Stratification based on serum proteomic profile could be of use for a better clinical classification of SSc patients, adding new insights to the underlying pathophysiological mechanisms.2) Combination of disease classifying autoantibodies with principal pathophysiological processes and serum proteomic profiles can help to elucidate and strengthen the diagnosis as well as the prognosis in SSC.AcknowledgementsSupported by ISCIII (RICOR-RD21/0002/0033) co-financed with FEDER.Disclosure of InterestsNone declared.

Factor Inhibiting the Hypoxia Inducible Factor (FIH) is an oxygen-dependent asparaginyl hydroxylase that plays a role in the cellular response to changes in oxygen concentrations. It was first found to catalyse the post translational hydroxylation of a family of transcription factors known as Hypoxia Inducible Factors (HIFs), and thus acts as a cellular sensor of hypoxia. Subsequently, other protein substrates were discovered containing ankyrin (ANK) repeat domains, although their activities were not affected by hydroxylation. FIH null mice display a metabolic phenotype which is not obviously linked to HIF regulation, supporting the existence of other important substrates of FIH. Hence the search for novel substrates of FIH was pursued, with particular interest in target proteins that upon modification by FIH would have a functional cellular outcome. Methods such as yeast two hybrid and pull down assays were previously employed to discover new substrates of FIH, but this limited searches to proteins with a relatively strong affinity for FIH. In research by a collaborator, a non-biased bioinformatic search was used to identify novel potential FIH substrates. Interestingly, the search identified a family of 5 ANK proteins present in the Orf virus (ORFV) as likely substrates of FIH. Preliminary experiments in our laboratory showed that they could interact with human FIH (hFIH), and some showed activity in CO₂ capture assays, consistent with hydroxylation. The first aim of this thesis was to determine whether the 5 ORFV ANK proteins, 008, 123, 126, 128 and 129 were substrates of FIH. Given that ORFV is an ovine virus, ovine FIH (oFIH) was cloned and purified, with recombinant protein displaying similar activity to recombinant hFIH. The ORFV ANK domains were cloned, expressed in bacteria and purified for analysis with recombinant oFIH. The hydroxylation state of these proteins was first analysed indirectly using in vitro hydroxylation assay and 008, 126 and 129 displayed high activity, consistent with being effectively hydroxylated by FIH, whereas 123 and 128 displayed low activity. Mass spectrometry (MS) of the recombinant proteins confirmed FIH-mediated hydroxylation of N40 in 008, N285 in 126, and N44 in 129, with poor ionisation preventing definitive conclusions regarding hydroxylation of 123 and 128. The ANK proteins of another closely related poxvirus, vaccinia virus (VACV), were also predicted to be substrates of FIH, but analysis of recombinant VACV ANK proteins showed no evidence of hydroxylation. These data indicate that these hydroxylation events are not conserved across the poxvirus family. The second aim was to characterise the functional role for this interaction between ORFV ANK proteins and FIH. Given the poorly characterised roles of the ORFV ANK proteins, and the relatively stable interaction between these proteins and FIH, these studies focused on the hypothesis that the ORFV ANK proteins could sequester FIH and subsequently upregulate HIF activity upon viral infection. Cell-based reporter gene assays confirmed that expression of the ORFV ANKs could sequester FIH and upregulate the HIFα transactivation domain, leading to increased HIF activity. Further supporting this hypothesis, ORFV infected cells that overexpressed or were deficient in FIH showed the induction of well characterised HIF target genes in a FIH-dependent manner. These data identified a novel mechanism of viral induced modulation of HIF activity, via the sequestration of FIH. The final aim of this thesis was to analyse OTUB1, a non-ANK and non-HIF protein discovered by our collaborators as a putative novel substrate of FIH. The hydroxylation of OTUB1 on N22 by FIH in vitro was confirmed using synthetic OTUB1 peptides in CO₂ capture assays.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018