Ubiquitination Pattern Research Articles

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development.

The ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that the ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of the ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.

The effect of hormones insulin and glucagon on ubiquitin modifications elucidated by proteomics in liver cells

AimsInsulin action is intertwined with changing levels of glucose and counter-regulatory hormone glucagon. While insulin lowers blood sugar level, glucagon raises it by promoting the breakdown of the stored glycogen in liver and releases glucose into the bloodstream. The hormones insulin and glucagon are key in the pathogenesis of type 2 diabetes (T2D). Insulin resistance is a primary predisposing factor for diabetes. Phosphorylation of insulin signaling molecules is altered in the insulin-resistant state. However, ubiquitin (Ub) modifications in insulin-resistant state are relatively understudied. To dissect the underlying mechanisms, we performed a proteomics study on hepatoma cells to study the regulation of ubiquitination by insulin and glucagon. Materials and methodsWe performed western blotting, immunoprecipitations, and affinity pull down using tandem Ub binding entities (TUBE) reagents on hepatoma cells treated with insulin or glucagon. Next, we performed MS/MS analysis on Ub-linkage specific affinity pull down samples. Gene ontology analysis and protein-protein interaction network analysis was performed using DAVID GO and STRING db, respectively. Key findingsThe ubiquitination pattern of total Ub, K48-linked Ub, and K63-linked Ub was altered with the treatment of hormones insulin and glucagon. Ubiquitination in immunoprecipitated samples showed enrichment with total Ub and K48-linked Ub but not with K63-linked Ub. Ubiquitination by treatment with hormones mainly enriched key signaling pathways MAPK, Akt, oxidative stress etc. SignificanceOur study identified key altered proteins and signal transduction pathways which aids in understanding the mechanisms of hormonal action on ubiquitination and identify new therapeutic targets for T2D.

Abstract 2567: Exploring novel roles of the GPCR Smoothened in AKT signaling and drug resistance

Abstract The Smoothened receptor (SMO) belongs to the frizzled-class-G-protein coupled receptor and its activity can be classified in the canonical and the non-canonical pathway. The canonical pathway concludes in the transcriptional activation of the Hedgehog target genes through the GLI family of transcription factors whereas the non-canonical pathway leads to the recruitment and activation of trimeric G proteins. Recently, we have reported two novel roles of SMO with relevance to cell survival and drug resistance in DLBCL. In the first, SMO promotes a shift in the ubiquitination pattern for TNF receptor-associated factor 6 (TRAF6) from degradation inducing K48-ubiquitin branching towards K63-ubiquitination. TRAF6 is a K63-Ub E3 ubiquitin ligase for AKT and K63-ubiquitination of AKT increases membrane recruitment, activation and stability. Evidently, SMO promotes the formation of a scaffold-like protein complex including the de-ubiquitinating enzyme Ubiquitin specific peptidase 8 (USP8) and TRAF6. In its second novel role, SMO influences the vesicular sorting of IGF1R and other receptor tyrosine kinases. Loss of SMO leads to increased IGF1R lysosomal degradation and reduced IGF1R recycling, while overexpression increases IGF1R levels and AKT signaling. These observations indicate SMO receptor biology outside of the established canonical and non-canonical pathway functions. We have set out to use BioID to investigate the SMO interactome that contributes to these novel functionalities using a fusion protein of SMO and the reengineered E. coli biotinyl transferase BirA (Turbo) as bait. Preliminary BioID using SMO-Turbo returned a list of candidates which will require further data filtering approaches, but thus far shows a marked enrichment for proteins involved in vesicular trafficking. Citation Format: Samantha Manuel, Nitin Agarwal, Daniel Bilbao, Francisco Vega, Ralf Landgraf. Exploring novel roles of the GPCR Smoothened in AKT signaling and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2567.

Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery

Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research.

Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5.

Mitochondrial homeostasis is tightly controlled by ubiquitination. The mitochondrial integral membrane ubiquitin ligase MARCH5 is a crucial regulator of mitochondrial membrane fission, fusion, and disposal through mitophagy. In addition, the lipid composition of mitochondrial membranes can determine mitochondrial dynamics and organelle turnover. However, how lipids influence the ubiquitination processes that control mitochondrial homeostasis remains unknown. Here, we show that lipids common to the mitochondrial membranes interact with MARCH5 and affect its activity and stability depending on the lipid composition in vitro. As the only one of the tested lipids, cardiolipin binding to purified MARCH5 induces a significant decrease in thermal stability, whereas stabilisation increases the strongest in the presence of phosphatidic acid. Furthermore, we observe that the addition of lipids to purified MARCH5 alters the ubiquitination pattern. Specifically, cardiolipin enhances auto-ubiquitination of MARCH5. Our work shows that lipids can directly affect the activity of ubiquitin ligases and suggests that the lipid composition in mitochondrial membranes could control ubiquitination-dependent mechanisms that regulate the dynamics and turnover of mitochondria.

Clonal Cytogenetic Abnormalities in Myeloid Neoplasms Arising from Fanconi Anemia — a Single Center Study from India

Fanconi anemia (FA) is a genomic instability disease caused by defective DNA repair which predisposes patients into developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Identification of clonal abnormalities in FA patients is vital to detect transformation before the onset of a high-grade MDS/leukemia.We analyzed bone marrow (BM) samples from a series of 50 patients with FA who were diagnosed at the Christian Medical College, Vellore, India from January 2005 - July 2017 by peripheral chromosome breakage analysis (CBA) or FANCD2 ubiquitination analysis of peripheral blood or dermal fibroblasts. Inclusion in the current study was based on the availability of karyotyping samples performed on the BM aspirate collected at diagnosis. Peripheral blood and dermal fibroblasts were collected at diagnosis for CBA and FANCD2 western blot.All 50 patients evaluated had phenotypic features suggestive of FA. There were 28 patients who had increased CBA scores alone while 21 had both increased CBA scores as well as defective FANCD2 ubiquitination. One patient with negative CBA score showing hematopoietic mosaicism with defective FANCD2 ubiquitination pattern in dermal fibroblasts alone was also included in the study.The median age at diagnosis was 16 years (range 1-35) and 35 (70%) were male. Abnormal karyotypes were obtained in 38 patients while the remaining (n=12) showed a normal karyotype. Examination of bone marrow aspirate and trephine was consistent with MDS in 29 (MDS-RS-MLD n= 3; MDS-EB n= 10; MDS-U n=12; h-MDS n=4), while the remaining had either AML (n=8) or a hypoplastic marrow (n=13). The 12 patients who had a normal karyotype were predominantly hypoplastic (n=8) or had either MDS (n=3) or AML (n=1). Abnormalities of chromosomes 1 and 7 were the most commonly reported chromosome aberration and was seen in 17 patients each (45%). A partial deletion 5q was present in 7 (18%) patients while gains of chromosomes 8 and 21 were seen in 5 and 6 patients respectively. Complex karyotypes involving abnormalities of 3 or more chromosomes were seen in 16 (42%) patients who had MDS/AML. FA gene mutation analysis of 8 patients showed FANCA gene mutations in 7 patients (4-abnormal KT, 3-normal KT) while 1 patient with abnormal karyotype had a mutation in the FANCJ gene.Post treatment karyotypes were available in 2 patients with AML and 1 patient with MDS which showed persistence of the leukemic clone identified at diagnosis (n=1) or clonal evolution (n=1, gain of chromosome 9) while the third patient showed a new clone after treatment which retained two of the three abnormalities documented at diagnosis.Using the Revised International Prognostic Scoring system for MDS majority of the patients (n=20) with MDS were classified in the poor or very poor risk groups while the remaining patients were in the good (n=6) or intermediate (n=3) risk group.Five of the 8 patients with AML were classified as poor risk according to the refined MRC cytogenetic classification while 3 were grouped in the intermediate risk group.Among the 13 patients showing a hypoplastic marrow, one patient had monosomy 7 which has been shown to have an increased risk of transformation to MDS/AML. One patient who had gain of 1q due to an unbalanced translocation transformed to AML in 9 months.We report a distinct pattern of chromosome abnormalities in patients with FA-MDS/AML consistent with previous reports, underscoring the need for inclusion of karyotype analysis in the laboratory evaluation strategy of FA patients for early detection of transformation to hematologic malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Herpes simplex virus 1 infection induces ubiquitination of UBE1a.

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

RIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.

On the structure and function of Sorghum bicolor CHIP (carboxyl terminus of Hsc70-interacting protein): A link between chaperone and proteasome systems

The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants.

Semisynthetic and Enzyme‐Mediated Conjugate Preparations Illuminate the Ubiquitination‐Dependent Aggregation of Tau Protein

AbstractIn the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination‐linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four‐repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site‐specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme‐derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post‐translational modification on aberrant protein self‐assembly.

UBE3Aren substratuak identifikatzen Angelman sindromearen ingurukoak argitzeko

Angelman sindromea neurogarapenezko gaixotasun bakana da, eta, arrazoiak arrazoi, neuronetan amarengandik jasotako UBE3A genearen kopia adierazten ez duten gizabanakoek pairatzen dute. UBE3Ak E3 ubikuitina ligasa entzima bat kodetzen du, zeina, E1 eta E2 entzimekin elkarlanean, itu-proteinei ubikuitina deritzon molekula txiki bat itsasteaz arduratzen den. Proteina bati ubikuitina gehitzeak berebiziko eragina izan dezake beraren patuan; izan ere, besteak beste, proteinen biziraupena, zelula barruko kokapena edota proteinen arteko elkarrekintzak baldintzatu ditzake. Beraz, ubikuitinazio-prozesuaren garrantzia ikusirik, eta UBE3Aren funtzioa zein den aintzat hartuta, pentsatzekoa da UBE3Aren faltan beraren itu-proteinen ubikuitinazio-maila gutxitua egongo dela, eta horrek eragin zuzena izango duela ez soilik proteinetan, baita zelularen funtzionamenduan ere. Angelman sindromea pairatzen dutenen zelula mailako kalteen jatorria ulertzeko, ezinbestekoa da UBE3Ak kodetzen duen E3 ubikuitina ligasaren itu-proteinak edo substratuak zeintzuk diren jakitea. Hori argitze aldera, UBE3A gainadierazten duten eta gainadierazten ez duten Drosophila eulietan ubikuitinatuta dauden proteinak aztertu eta konparatu ditugu. Ubikuitinatutako proteinak arrantzatzeko, gure laborategian garatutako BioUb estrategia erabili dugu, eta, ondoren, horiek identifikatzeko eta kuantifikatzeko, masa-espektrometria (MS) erabili dugu. Identifikatutako ehunka proteinetatik, 79k betetzen dituzte UBE3Aren substratu izateko irizpideak. Horien artean dago Rnp10, aurretiaz UBE3Aren itu-proteina gisa deskribatu den proteina. Horrez gain, Rngo deituriko proteinak piztu zuen gure arreta, eta, biologia molekularreko teknika osagarriak erabiliz, MS bidezko emaitzak berretsi ditugu. Gainera, giza zelulak erabiliz, Rngo-ren homologoa den DDI1 ere UBE3Aren substratua dela frogatu dugu. Etorkizunean, gure helburu nagusiak hiru dira: (i) MS bidez identifikatutako UBE3Aren substratu-izangai gehiago egiaztatzea, (ii) proteina horiek gizakietan ere UBE3Aren substratuak direnentz frogatzea, eta (iii) UBE3Aren substratuek zelula mailan betetzen dituzten funtzioak aztertzea.

192-OR: A CRISPR Screen Identifies Rfwd2 as a Regulator of Brown Fat Glucose Transport

Brown adipose tissue (BAT) generates heat to regulate body temperature, and to support this energy expending activity it takes up glucose from circulation. Cold exposure regimens that activate BAT improve insulin sensitivity, presumably by decreasing circulating glucose levels. Interestingly, glucose uptake during cold is specific to BAT and occurs at low circulating insulin levels, suggesting alternative pathways that do not exist in other insulin sensitive tissues may regulate brown adipocyte glucose uptake. To identify putative regulators of brown adipocyte glucose uptake, we performed a whole genome CRISPR screen using the uptake of the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) as the readout on mouse brown adipocytes differentiated in vitro. By sorting single brown adipocytes according to their fluorescence, we separated the top 5%, bottom 5% and the remainder of cells using fluorescence activated cell sorting (FACS) and then sequenced to identify genes acting as both putative positive and negative regulators of glucose uptake. Genes identified as potential hits could be classified by gene ontology and belonged mostly to groups related to metabolism and energy homeostasis. A list of 25 potential regulatory genes were validated using the same 2-NBDG strategy used to identify them as well as radiolabeled glucose uptake in vitro. From these genes, 5 were further validated using a second CRISPR based strategy to block gene function. One gene that we identified is the E3 ubiquitin ligase Rfwd2, which is highly expressed in BAT and is known to regulate lipid metabolism. When Rfwd2 was deleted from brown adipocytes the global ubiquitination pattern was changed and glucose uptake was increased in both the basal and insulin stimulated states. These data suggest that targets of this ligase may play a role in the cell-signaling cascade that activates glucose uptake and these targets could further regulate BAT glucose uptake in vivo. Disclosure M. Lynes: None. Q. Huang: None. P. Yi: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd. Funding National Institutes of Health (P30DK036836, R01DK077097, R01DK102898, F32DK102320, K01DK111714)

Inhibition of apoptosis via CHIP-mediated proteasomal degradation of TAp73α.

TAp73, a homologous of tumor suppressor p53, regulates apoptosis in a p53-independent manner and its suppressive as well as stimulatory role in promoting angiogenesis has been reported. It exists in multiple isoforms which varies structurally in their N-terminus and C-terminus region and crucial interplay among them guides the decision of cell survival and death. As molecular chaperones control both stability and degradation of TAp73, selective regulation of p73 isoforms has implication upon developing new therapeutic for hypoxic tumor. We have discovered that under DNA damage carboxy terminus Hsp70 interacting protein (CHIP's) antiapoptotic function is displayed via its E3 ligase activity that inhibits exclusively TAp73α-mediated apoptosis in cancer cell. The decrease in TAp73α level by CHIP as it is supported by increased ubiquitination pattern is reverted back by sh-CHIP. Further, the transactivation of p53-downstream apoptotic genes BAX, PUMA and PIG3 by TAp73α is also shown to be subsequently inhibited by CHIP. The tetratricopeptide TPR-domain of CHIP in its amino-terminus interacts with the carboxy-terminus of TAp73α and ΔNp73α and as a result, U-BOX domain of CHIP in the carboxy-terminus is able to ubiquitinate TAp73α for proteasomal degradation. Due to lack of C-terminus in TAp73β, CHIP fails to interact with and degrade it. In conclusion, we have thus uncovered for the first time a novel mechanism of chaperone-assisted regulation of p73 stability as well as its apoptotic functions by CHIP that might be utilized to develop new anticancer strategies.

Abstract B16: The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells

Abstract Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to study the functions of nIGF-1R in human embryonic stem cells (hESCs) and in the IGF-1R knockout (igf1r -/-) (R-) and the R- cells overexpressing human IGF1R (R+) mouse embryonic fibroblasts (MEFs), using a mass-spectrometry based approach. Methods and Results: Using subcellular protein fractionation (Qiagen Qproteome Cell Compartment Kit, Qiagen, Germany) and western blot (WB), as well as immunofluorescence, we detected the expression of the nIGF-1R in hESCs, human fibroblasts, MEFs, and in cancer cell lines. To investigate potential functions of nIGF-1R in hESCs, nIGF-1R binding partners were identified using co-immunoprecipitation (Co-IP) and mass spectrometry (MS) (hybrid LTQ-Orbitrap Velos mass spectrometer, Thermo Scientific). Multiple protein candidates were identified including PCNA. IGF-1R-PCNA association was confirmed by Co-IP/immunoblot and by in situ proximity ligation assay (PLA) (OLINK Bioscience, Uppsala, Sweden) to morphologically demonstrate the nuclear localization of the complex. The association of nIGF-1R with PCNA could only be detected in hESCs, MEFs and in human fibroblast cell lines, but not in any of the seven cancer cell lines used. Using a phospho-tyrosine antibody we detected the phosphorylation of PCNA immunoprecipitated from hESCs and R+ but not from R- MEFs or cancer cell lines. Using an in vitro kinase assay, recombinant IGF-1R kinase domain and His-tag recombinant PCNA, we showed that IGF-1R directly phosphorylates PCNA. Using LC-MS/MS and site directed mutagenesis, 3 tyrosine phosphorylation sites (Y60,133,250) were identified. Although PCNA ubiquitination was detected by WB of PCNA immunoprecipitated from R+ MEFs, PCNA protein stability was not affected. The ubiquitination pattern resembled the K63-linked polyubiquitination. Using Co-IP/WB, Ubc13 and SHPRH/HLTF were found to be associated with PCNA in R+ but not in R- cells. Similar results were also achieved for the mono-ubiquitin Rad18 E3 ligase and the main TLS polymerase DNA polymerase. Results were confirmed in hESCs but not in cancer cell lines. Further, PCNA ubiquitination was increased after DNA damage using UV irradiation in R+ MEFs and DNA replication was delayed after methyl methanesulfonate (MMS) treatment, while replication fork stalling in R- cells was highly significant (DNA-fiber labeling assay). Inhibition or knockout of IGF-1R abrogated PCNA phosphorylation, ubiquitination, progress through S-phase of cell cycle, or restart of DNA synthesis after inducing DNA damage. Conclusions: Our results demonstrate a novel role of nIGF-1R in regulating the DNA damage tolerance pathway through PCNA phosphorylation and ubiquitination, potentially maintaining genomic stability in in hESC and in normal fibroblasts. In contrast, nIGF-1R does not interact with PCNA in cancer cell lines. Citation Format: Ahmed Waraky, Yingbo Lin, Eiman Aleem, Olle Larsson. The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B16.

Abstract A81: Investigation of possible cell state dependent differential protein homeostasis mechanism(s)

Abstract Proteasome inhibition has been shown to be an important strategy for treating many types of cancer. Although proteasome inhibitors are effective at eradicating rapidly proliferating cancer cells, a subset of cancer cells enter quiescence and survive, contributing to tumor recurrence. Using primary human fibroblasts as a model system we have shown that quiescent cells have decreased reactive oxygen species levels and protein aggregates, and undergo apoptosis less frequently than proliferating cells in response to proteasome inhibition. However, the precise mechanism by which quiescent cells become resistant to proteasome inhibition remains largely unknown. Using a GFP-tagged proteasome reporter, we investigated mechanisms protecting quiescent cells from proteasome inhibition-induced apoptosis. We found that, in proliferating cells, proteasome inhibition by MG132 led to high levels of a GFP-tagged proteasome reporter with a visible ubiquitination pattern. In contrast, MG132 treatment of quiescent cells resulted in reduced levels of the ubiquitinated form of this reporter as observed by Western blot analysis. Interestingly, preliminary immunoprecipitation followed by mass-spectrometry analysis revealed none canonical ubiquitin modification on lysine 27 of the reporter in quiescent cells. These findings suggested that quiescent cells may have one or more alternative pathways preventing specific proteasome substrates accumulation in response to proteasome inhibition. Moreover, our data has shown that, upon proteasome inhibition, a GFP-tagged proteasome reporter was secreted into the condition medium. Secretion of cytoplasmic proteins in response to proteasome inhibition is one possible mechanism for reducing protein aggregation and cytotoxicity. Identification of regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in cancer treatment. Citation Format: Gordon-Victor Fon, Chandler R. Keller, David H. Perlman, Aster Legesse-Miller. Investigation of possible cell state dependent differential protein homeostasis mechanism(s). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A81.

Low-density lipoprotein receptor-related protein 1 mediates α1-antitrypsin internalization in CD4+ T lymphocytes.

In α1-antitrypsin-deficient HIV patients, an accelerated decline of CD4(+) T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4(+) T cells. Thus, primary CD4(+) T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4(+) T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4(+) T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4(+) T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1-mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome-lysosome-cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.

Detection of ubiquitinated huntingtin species in intracellular aggregates.

Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

Platelet FcγRIIA Signaling Results in Ubiquitination and Cellular Translocation of Activated Syk

Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunotyrosine activation motif (ITAM) signaling, such as FcγRIIA, GPVI and CLEC-2. We previously reported Syk ubiquitination following GPVI stimulation, and the role of c-cbl as the E3 ubiquitin ligase (Dangelmaier, Blood 2005). Ubiquitination is an important post-translational modification that modulates signal transduction by regulating the activity, subcellular localization or stability of proteins. We tested the hypothesis that ubiquitination participates in signaling, and examined ubiquitination of Syk downstream of platelet FcγRIIA activation.Using both washed human platelets and HEL cells, we observed ubiquitination of Syk upon FcγRIIA engagement by cross-linking IV.3 mAb (10 ug/ml) with goat anti-mouse Fab’2 (30 ug/ml). Both tyrosine phosphorylation and ubiquitination of Syk occurred within 15 sec, peaked by 1-3 min and decreased thereafter. The pattern of ubiquitination was consistent with 1 to 3 Ub molecules per Syk molecule. Ubiquitinated-Syk (Ub-Syk) was increased in the presence of PR-619, a deubiquitinating enzyme inhibitor, confirming ubiquitination of Syk. Ub-Syk associates with the cytoskeletal-rich platelet fraction, membrane skeleton fraction and with cytosolic fraction in detergent lysed platelets that were fractionated by lower g-forces (15,500 x g) and higher g-forces (100,000 x g). This suggests that Ub-Syk is translocated into all cellular compartments upon platelet activation. Ub-Syk was absent upon pre-treatment with Src-family kinase inhibitor PP2 (10 uM), but minimally affected in the presence of Syk inhibitor PRT318 (1 uM), in both platelets and HEL cells, as compared to DMSO treated control cells. Further, phosphorylation of c-cbl was inhibited strongly by PP2, but only slightly inhibited by PRT318, suggesting that ubiquitination of Syk depends on Src kinase activity. Of note, Ub-Syk was not degraded by the proteasome, since no accumulation of Ub-Syk was observed by pretreatment with proteasome inhibitors MG132 or Epoxomicin in either platelets or HEL cells, compared to control cells.In conclusion, Syk is ubiquitinated upon cross-linking platelet FcγRIIA and is translocated to all major subcellular compartments. Since ubiquitinated Syk is activation-dependent and not subject to proteasomal degradation, it likely serves as a novel adapter molecule for protein-protein interactions in mediating platelet activation via FcγRIIA. DisclosuresNo relevant conflicts of interest to declare.

Clinical and Molecular Characterization of Fanconi Anemia: An Indian Perspective

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by a defect in one of the DNA repair pathways. FA exhibits a high degree of clinical heterogeneity and the exact molecular basis of the difference in the phenotypes has not been well understood. Study of this disease in geographic regions with high consanguinity will provide valuable insights in genotype-phenotype correlation and the genetic and environmental modifiers of this disease. We carried out a comprehensive clinical and molecular analysis in 101 patients with pancytopenia, with age <20 years, seen at the department of Haematology, Christian Medical College, India between 2009 and 2014. Seventy six patients had characteristic physical abnormalities of FA, of which perioral hyperpigmentation (42%) was the most common in these patients. We also included 25 patients with aplastic anemia as controls. The median age at diagnosis was 11 years (range 4-30) and sex ratio between males to females was 3:2. Chromosome breakage analysis (CBA) was performed on peripheral blood samples at diagnosis. Forty metaphases each from patient and normal control were assessed for sensitivity to Mitomycin C as per the protocol published in Mayo Clinic Proceedings (1997). CBA score greater than the cut off value of 40 was seen in 63% (n=48/76) of the patients. However, 23% (n=18/76) had ambiguous or borderline score (score 30-40; n= 12, score 20-30; n=4, score <10-20; n=2) and 13% (n=10/76) of patients were not sensitive to Mitomycin C. The control group did not display sensitivity or had mild degree of breakage (CBA score: 4.1-20). FANCD2 ubiquitination analysis of these patients with physical abnormalities in peripheral blood and fibroblasts showed absence of ubiquitination in 69 (90%) of the patients while the control group had normal ubiquitination pattern. To evaluate mosaicism, both peripheral blood and fibroblast samples were analyzed in 59 patients and FANCD2 ubiquitination was defective in both tissues in 55 (93%) patients. In 3 patients, FANCD2 ubiquitination was defective in fibroblasts, but not in peripheral blood indicative of mosaicism of this disease in these patients. Three patients had a normal ubiquitination pattern but had high CBA score and this might be due to mutations in any of the genes that function in the pathway downstream of FANCD2 ubiquitination and requires further evaluation. Four patients who had physical features suggestive of FA were negative by both CBA and FANCD2 western blot. In the control group, both peripheral blood and fibroblast samples were available in 19 patients all of which showed normal FANCD2 ubiquitination. In order to further characterize the FA subtypes, we generated lentiviral vectors to express FANCA, FANCC and FANCG genes for complementation assay. Fibroblasts from 20 patients were transduced with lentiviral FANCA and in 17 (85%) FANCD2 ubiquitination could be restored suggesting a high frequency of FANCA defects in Indian population. Mutation screening of FANCA in a subset of patients showed large deletions in five of which three are novel (ex 10_37 del,ex 6_14 del and ex 1_4 del). There was a deletion of two nucleotides resulting in a frameshift mutation in one patient (c.3760_3761delGA) and a missense mutation (c.2786A>C) in another. Our data suggests that FANCD2 ubiquitination analysis in conjunction with CBA is useful for the diagnosis of FA and detection of mosaicism and the complementation analysis shows high frequency of FANCA defects in patients with FA in Indian population. DisclosuresNo relevant conflicts of interest to declare.