Ubiquitination Activity Research Articles

Small-molecule inhibitors of linear ubiquitin chain assembly complex (LUBAC), HOIPINs, suppress NF-κB signaling

Nuclear factor-κB (NF-κB) is a crucial transcription factor family involved in the regulation of immune and inflammatory responses and cell survival. The linear ubiquitin chain assembly complex (LUBAC), composed of the HOIL-1L, HOIP, and SHARPIN subunits, specifically generates Met1-linked linear ubiquitin chains through the ubiquitin ligase activity in HOIP, and activates the NF-κB pathway. We recently identified a chemical inhibitor of LUBAC, which we named HOIPIN-1 (HOIP inhibitor-1). To improve the potency of HOIPIN-1, we synthesized 7 derivatives (HOIPIN-2∼8), and analyzed their effects on LUBAC and NF-κB activation. Among them, HOIPIN-8 suppressed the linear ubiquitination activity by recombinant LUBAC at an IC50 value of 11 nM, corresponding to a 255-fold increase over that of HOIPIN-1. Furthermore, as compared with HOIPIN-1, HOIPIN-8 showed 10-fold and 4-fold enhanced inhibitory activities on LUBAC- and TNF-α-induced NF-κB activation respectively, without cytotoxicity. These results indicated that HOIPIN-8 is a powerful tool to explore the physiological functions of LUBAC.

SU103 - UBIQUITINATED PROTEINS ARE ELEVATED IN THE BLOOD AND BRAIN OF INDIVIDUALS WITH SCHIZOPHRENIA

Background Evidence from genome-wide association, microarray, and protein studies have indicated dysregulation of genes and proteins within the Ubiquitin Proteasome Pathway (UPP) in the blood and brain of individuals with schizophrenia. However, it is not clear which components of the UPP, if any, are dysregulated in both blood and brain. Methods We obtained frozen postmortem Orbitofrontal Cortex (OFC) tissue from 76 (38 schizophrenia, 38 control) individuals and erythrocytes from 181 participants, comprising 30 individuals with recent onset schizophrenia (mean illness duration=1 year), 63 individuals with schizophrenia treated with clozapine and considered ‘treatment-resistant’ (mean illness duration=17 years), and 88 control participants. Free ubiquitin and ubiquitinated protein levels as well as endogenous ubiquitination and proteasome activity were quantified in erythrocytes and postmortem OFC blind to diagnosis. Results Individuals with schizophrenia had higher levels of ubiquitinated proteins compared to those with recent onset schizophrenia (p Discussion Our results suggest that protein homeostasis may be abnormal in both the blood and brain of those with schizophrenia, particularly in the later stages or specific sub-groups of the illness. Follow-up studies to determine the mechanism by which this abnormality in protein homeostasis arises and whether its presence in blood has prognostic utility are warranted.

Purification and functional characterization of the DUB domain of SdeA.

Intracellular pathogens like Legionella pneumophila hijack the host ubiquitination network in order to create a facultative niche for their survival by means of effector molecules secreted into the host cell. Some of these effectors function as ubiquitin ligases or deubiquitinases, among other types of enzymes. Deubiquitinating enzymes (DUBs) remove ubiquitin or ubiquitin-like modifiers from conjugated substrates to regulate various cellular processes. Members of the SidE effector family from the L. pneumophila pathogen harbor multiple functional domains that possess discrete biochemical activities impinging on host ubiquitin signaling. At the N-terminal end of these ~1500-residue proteins is a ~200-residue conserved DUB domain capable of recognizing both ubiquitin and the NEDD8 Ubl. SdeA, a member of the SidE family, plays an important role in intracellular bacterial replication. Downstream domains in this protein also catalyze substrate ubiquitination via a phosphoribosyl linkage. Several mammalian Rab proteins (Rab1, Rab30, and Rab33) have been shown to be targeted. The novel mechanism is independent of the classical E1 and E2 ubiquitin ligation machinery and does not require ATP. The N-terminal DUB domain, which does not appear to affect this ubiquitination activity, but it catalyzes cleavage of three different types of polyubiquitination chains (K11, K48, and K63) commonly found in host cells. This chapter describes methods, including purification of recombinant SdeA (full-length and DUB domain alone), and enzymatic assays that have been utilized to characterize the deubiquitination activity of SdeA.

Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE.

Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).

Methods to analyze STUbL activity.

Posttranslational modification with small ubiquitin-like modifier (SUMO) plays an important role in many biological processes. SUMO-targeted ubiquitin E3 ligases (STUbLs) are part of the really interesting new gene (RING)-type family of ubiquitin E3 ligases. STUbLs recognize their SUMO-modified substrates via SUMO-interaction motifs and ubiquitinate them via the RING domain. As a result, they form a link between the ubiquitin and SUMO signaling pathways. STUbL activity is required for the maintenance of genome stability, the repair of damaged DNA and to target SUMO-modified proteins for degradation by the proteasome. In vitro assays for STUbL activity have been developed and used to identify their cognate ubiquitin-conjugating enzymes (E2s), to determine their substrate requirements, and to characterize the types of ubiquitin chains linked to substrates. While we have focused on the STUbL RING finger protein 4 (RNF4) the methods we describe can be extended to other STUbLs. We also describe an assay for RNF4 ubiquitination activity based on fluorescence polarization, suitable for high-throughput compound screening in drug discovery.

Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7.

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase–target pair deemed unsuitable: the von Hippel–Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure–activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target–ligase combinations.

Distinct subcellular changes in proteasome activity and linkage-specific protein polyubiquitination in the amygdala during the consolidation and reconsolidation of a fear memory.

Numerous studies have supported a critical role for the ubiquitin-proteasome system (UPS) in the memory consolidation and reconsolidation processes. The protein targets and functional role of ubiquitin-proteasome activity can vary widely across cellular compartments, however, it is unknown how UPS activity changes within the nuclear, cytoplasmic, and synaptic regions in response to learning or memory retrieval. Additionally, while previous studies have focused on degradation-specific protein polyubiquitination, it is unknown how learning alters other polyubiquitin tags that are not targeted by the proteasome. Using cellular fractionation protocols in combination with linkage-specific polyubiquitin antibodies, we examined subcellular changes in ubiquitin-proteasome activity in the amygdala during memory consolidation and reconsolidation. Following memory acquisition, overall protein ubiquitination and proteasome activity simultaneously increased in the nucleus and decreased in the synaptic and cytoplasmic regions. The nuclear increases were associated with upregulation of degradation-specific (K48) and degradation-independent (K63, M1) polyubiquitin tags, suggesting multiple functions for ubiquitin signaling within this region. Interestingly, retrieval induced a very different pattern of ubiquitin-proteasome activity in the amygdala, consisting of increases in overall protein ubiquitination and proteasome activity and K48-, K63-, and M1-polyubiquitin tags in the synaptic, but not nuclear or cytoplasmic regions. Collectively, learning and memory retrieval dynamically and differentially alter degradation-dependent and degradation-independent ubiquitin-proteasome activity across different cellular compartments, suggesting that the UPS may serve unique functions during memory consolidation and reconsolidation.

Legionella pneumophila inhibits immune signalling via MavC-mediated transglutaminase-induced ubiquitination of UBE2N.

SummaryThe bacterial pathogen Legionella pneumophila modulates host immunity using effectors translocated by its Dot/Icm transporter to facilitate its intracellular replication. A number of these effectors employ diverse mechanisms to interfere with protein ubiquitination, a posttranslational modification essential for immunity. Here we found that L. pneumophila induces monoubiquitination of the E2 enzyme UBE2N by its Dot/Icm substrate MavC(Lpg2147). We demonstrate that MavC is a transglutaminase that catalyzes covalent linkage of ubiquitin to Lys92 and Lys94 of UBE2N via Gln40. Similar to canonical transglutaminases, MavC possess deamidase activity that targets ubiquitin at Gln40. We identified Cys74 as the catalytic residue for both ubiquitination and deamidation activities. Furthermore, ubiquitination of UBE2N by MavC abolishes its activity in the formation of K63-type polyubiquitin chains, which dampens NFκB signaling in the initial phase of bacterial infection. Our results reveal an unprecedented mechanism of modulating host immunity by modifying a key ubiquitination enzyme by ubiquitin transglutamination.

TRIM25 Binds RNA to Modulate Cellular Anti-viral Defense

TRIM25 is a multi-domain, RING-type E3 ubiquitin ligase of the tripartite motif family that has important roles in multiple RNA-dependent processes. In particular, TRIM25 functions as an effector of RIG-I and ZAP, which are innate immune sensors that recognize viral RNA and induce ubiquitin-dependent anti-viral response mechanisms. TRIM25 is reported to also bind RNA, but the molecular details of this interaction or its relevance to anti-viral defense have not been elucidated. Here, we characterize the RNA-binding activity of TRIM25 and find that the protein binds both single-stranded and double-stranded RNA. Multiple regions of TRIM25 contribute to this functionality, including the C-terminal SPRY domain and a lysine-rich motif in the linker segment connecting the SPRY and coiled-coil domains. RNA binding modulates TRIM25's ubiquitination activity in vitro, its localization in cells, and its anti-viral activity. Taken together with other studies, our results indicate that RNA binding by TRIM25 has at least three important functional consequences: by enhancing ubiquitination activity, either through allosteric effects or through clustering of multiple TRIM25 molecules; by modulating the multi-domain structure of the TRIM25 dimer, and thereby structural coupling of the SPRY and RBCC elements during the ubiquitination reaction; and by facilitating subcellular localization of the E3 ligase during virus infection.

Parkinson\u2019s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

BackgroundParkinson’s disease (PD)-associated E3 ubiquitin ligase Parkin is enriched at glutamatergic synapses, where it ubiquitinates multiple substrates, suggesting that its mutation/loss-of-function could contribute to the etiology of PD by disrupting excitatory neurotransmission. Here, we evaluate the impact of four common PD-associated Parkin point mutations (T240M, R275W, R334C, G430D) on glutamatergic synaptic function in hippocampal neurons.ResultsWe find that expression of these point mutants in cultured hippocampal neurons from Parkin-deficient and Parkin-null backgrounds alters NMDA and AMPA receptor-mediated currents and cell-surface levels and prevents the induction of long-term depression. Mechanistically, we demonstrate that Parkin regulates NMDA receptor trafficking through its ubiquitination of GluN1, and that all four mutants are impaired in this ubiquitinating activity. Furthermore, Parkin regulates synaptic AMPA receptor trafficking via its binding and retention of the postsynaptic scaffold Homer1, and all mutants are similarly impaired in this capacity.ConclusionOur findings demonstrate that pathogenic Parkin mutations disrupt glutamatergic synaptic transmission in hippocampal neurons by impeding NMDA and AMPA receptor trafficking. Such effects may contribute to the pathophysiology of PD in PARK2 patients.

Activation of NF-κB induced by TRIMCyp showing a discrepancy between owl monkey and northern pig-tailed macaque

TRIMCyp generated by retrotransposition of a cyclophilin A inserting into TRIM5 locus, has been identified in owl monkey and most of Old World monkeys (OWM). Owl monkey TRIMCyp (omTRIMCyp) inhibits HIV-1 infection by direct interaction with viral capsid and indirect innate immune induction, whereas most of TRIMCyps from OWM cannot inhibit HIV-1, and the impact of which on immunoregulation is largely unknown. Here we reported that omTRIMCyp induces NF-κB, AP-1 and IFN-β activation in a dose-dependent manner, while TRIMCyp from northern pig-tailed macaque (npmTRIMCyp) does not activate NF-κB and moderately enhances AP-1 and IFN-β activities. The Cyclophilin A (CypA) domain plays an important role in omTRIMCyp-mediated NF-κB activation, and RBCC domains have a synergetic effect. We further indicated the mechanism by which npmTRIMCyp unable to activate NF-κB is that npmTRIMCyp hardly phosphorylates IκBα, different from omTRIMCyp which dramatically induces IκBα phosphorylation. Ubiquitination activity of omTRIMCyp was greater than npmTRIMCyp, although both could be ubiquitylated. Given that npmTRIMCyp neither interacts with viral capsid resulting in susceptibility to HIV-1 infection, nor activates NF-κB that is indispensable to HIV-1 provirus transcription, we proposed a model that npmTRIMCyp may play an important role in HIV-1 infected northern pig-tailed macaque with latency.

TRIM17 and TRIM28 antagonistically regulate the ubiquitination and anti-apoptotic activity of BCL2A1.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.

β-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe~Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the β-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub.

Induction of DUSP14 ubiquitination by PRMT5-mediated arginine methylation.

Dual-specificity phosphatase (DUSP)14 (also known as MAP-kinase phosphatase 6) inhibits T-cell receptor (TCR) signaling and T-cell–mediated immune responses by inactivation of the TGF-β activated kinase 1 binding protein (TAB1)–TGF-β activated kinase 1 (TAK1) complex and ERK. DUSP14 phosphatase activity is induced by the E3 ligase TNF receptor associated factor (TRAF)2-mediated Lys63-linked ubiquitination. Here we report an interaction between DUSP14 and protein arginine methyltransferase (PRMT)5 by proximity ligation assay; similarly, DUSP14 directly interacted with TAB1 but not TAK1. DUSP14 is methylated by PRMT5 at arginine 17, 38, and 45 residues. The DUSP14 triple-methylation mutant was impaired in PRMT5-mediated arginine methylation, TRAF2-mediated lysine ubiquitination, and DUSP14 phosphatase activity. Consistently, DUSP14 methylation, TRAF2 binding, and DUSP14 ubiquitination were attenuated by PRMT5 short hairpin RNA knockdown. Furthermore, DUSP14 was inducibly interacted with PRMT5 and was methylated during TCR signaling in T cells. Together, these findings reveal a novel regulatory mechanism of DUSP14 by which PRMT5-mediated arginine methylation may sequentially stimulate TRAF2-mediated DUSP14 ubiquitination and phosphatase activity, leading to inhibition of TCR signaling.—Yang, C.-Y., Chiu, L.-L., Chang, C.-C., Chuang, H.-C., Tan, T.-H. Induction of DUSP14 ubiquitination by PRMT5-mediated arginine methylation.

Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling.

The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.

Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers.

Protein ubiquitination is involved in many cellular processes, such as protein degradation, DNA repair, and signal transduction pathways. Ubiquitin-conjugating (E2) enzymes of the ubiquitination pathway are associated with various cancers, such as leukemia, lung cancer, and gastric cancer. However, to date, detection of E2 activities is not practicable for capturing the pathological conditions of cancers due to complications related to the enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. Artificial RING fingers (ARFs) were developed to conveniently detect E2 activities during the ubiquitination reaction. ARFs were created by grafting the active sites of ubiquitin-ligating (E3) enzymes onto amino acid sequences with 38 residues. The grafting design downsized E3s to small molecules (ARFs). Such an ARF is a multifunctional molecule that possesses specific E2-binding capabilities and ubiquitinates itself without a substrate. In this review, we discuss the major findings from recent investigations on a new molecular design for ARFs and their simplified detection system for E2 activities. The use of the ARF allowed us to monitor E2 activities using acute promyelocytic leukemia (APL)-derived cells following treatment with the anticancer drug bortezomib. The molecular design of ARFs is extremely simple and convenient, and thus, may be a powerful tool for protein engineering. The ARF methodology may reveal a new screening method of E2s that will contribute to diagnostic techniques for cancers.

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

SummaryUbiquitination is a post-translational modification that regulates a myriad of cellular processes in eukaryotes1–4. The conventional ubiquitination cascade culminates in a covalent linkage between the C-terminus of ubiquitin (Ub) and a target protein, most often on a lysine sidechain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a novel mode of ubiquitination in which a phosphoribosylated ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6–8. To uncover the molecular mechanism of this unique post-translational modification, we determined the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the mART domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the PDE domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous PDE domain from the SidE family member SdeD9 in complex with Ub or ADPR-Ub. The structures suggest an intriguing mechanism for how SdeA processes ADPR-Ub to PR-Ub plus AMP and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-ubiquitination (PR-ubiquitination) and paves the way for future studies of this unusual type of ubiquitination in eukaryotes.

CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

Impact of altered phosphorylation on loss of function of juvenile Parkinsonism–associated genetic variants of the E3 ligase parkin

Autosomal recessive juvenile Parkinsonism (ARJP) is an inherited neurodegenerative disease in which 50% of affected individuals harbor mutations in the gene encoding the E3 ligase parkin. Parkin regulates the mitochondrial recycling pathway, which is induced by oxidative stress. In its native state, parkin is auto-inhibited by its N-terminal ubiquitin-like (Ubl) domain, which blocks the binding site for an incoming E2∼ubiquitin conjugate, needed for parkin's ubiquitination activity. Parkin is activated via phosphorylation of Ser-65 in its Ubl domain by PTEN-induced putative kinase 1 (PINK1) and a ubiquitin molecule phosphorylated at a position equivalent to Ser-65 in parkin. Here we have examined the underlying molecular mechanism of phosphorylation of parkin's Ubl domain carrying ARJP-associated substitutions and how altered phosphorylation modulates parkin activation and ubiquitination. We found that three substitutions in the Ubl domain (G12R, R33Q, and R42P) significantly decrease PINK1's ability to phosphorylate the Ubl domain. We noted that two basic loss-of-function substitutions (R33Q and R42P) are close to acidic patches in the proposed PINK1–parkin interface, indicating that ionic interactions at this site may be important for efficient parkin phosphorylation. Increased auto-ubiquitination with unique ubiquitin chain patterns was observed for two other Ubl domain substitutions (G12R and T55I), suggesting that these substitutions, along with phosphorylation, increase parkin degradation. Moreover, Ubl domain phosphorylation decreased its affinity for the potential effector protein ataxin-3, which edits ubiquitin chain building by parkin. Overall, our work provides a framework for the mechanisms of parkin's loss-of-function, indicating an interplay between ARJP-associated substitutions and phosphorylation of its Ubl domain.

The Misfolded Protein Client as a Determinant of Substrate Ubiquitination by the Hsp70/CHIP Complex

The E3 ubiquitin ligase CHIP (carboxy terminus of Hsc70 interacting protein) targets misfolded proteins chaperoned by heat shock protein 70 (Hsp70) for ubiquitination and subsequent proteasomal degradation. Hsp70 recruits and attempts to refold misfolded proteins, or “clients”, in iterative, ATP‐dependent cycles. In contrast, binding of Hsp70 to the tetratricopeptide repeat (TPR) domain of CHIP, allows for client proteins to be recruited for ubiquitination by CHIP. Ubiquitination of lysine residues on a client protein allows CHIP/Hsp70 complexes to promote the proteasomal degradation of the client. Thus, the CHIP/Hsp70 complex stands at the crossroads of degradative and refolding pathways of client proteins which is crucial for cellular protein quality control. The current knowledge in the field lacks a clear understanding of how the CHIP/Hsp70 complex executes its triage mechanism that directs misfolded proteins for either refolding or degradation. Recent models of ATP and ADP bound Hsp70 revealed the distances between the substrate binding domain (SBD) and the E2~ubiquitin conjugate active site as ~120 Å and ~60 Å respectively. This implies, with the dynamic conformational changes of Hsp70 complex, the ADP‐bound state of Hsp70 organizes the chaperoned ubiquitination complex by properly orienting the client with respect to CHIP which allows the client protein to span this 60 Å gap and accept the ubiquitin from the bound E2‐ubiquitin conjugate. Therefore, the distance between Hsp70 binding site and the active site of the E2~ubiquitin conjugate bound to CHIP plays a significant role as a determinant in the triage mechanism of CHIP/Hsp70. In this study, we focus on investigating the relationship between key distances within the CHIP/Hsp70/client complex and the ubiquitination activity of the CHIP/Hsp70 complex by utilizing in‐vitro ubiquitination assays of modified substrates.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.