The cullin-RING ubiquitin ligases (CRLs) are the largest family of multi-subunit E3 ligases in eukaryotes, which ubiquitinate protein substrates in numerous cellular pathways. CRLs share a common ...arched scaffold and a RING domain catalytic subunit, but use different adaptors and substrate receptors to assemble unique E3 machineries. In comparison to the first CRL structure, recent findings have revealed increased complexity in the overall architecture and assembly mode of CRLs, including multi-domain organization, inter-domain flexibility, and subunit dimerization. These features highlight the capacity of CRLs to catalyze protein ubiquitination under distinct cellular contexts and in response to diverse signals. As the first installment of a two-review series, this article will focus on recent advances in our understanding of CRL assembly mechanisms.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels ...are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.
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KISLJ, NUK, SBMB, UL, UM, UPUK
Antibody–drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a ...chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide–alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.
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IJS, KILJ, NUK, PNG, UL, UM
▶ Recent structural studies have revealed numerous ways in which CRL E3 ligase activities are controlled, including: ▶ Mechanisms of multimodal CRL E3 ligase activation by covalent attachment of the ...ubiquitin-like protein NEDD8. ▶ A novel ‘dual E3’ mechanism for ligation of NEDD8 to cullins. ▶ Mechanism inhibition of CRL assembly/activity by CAND1. ▶ Numerous mechanisms of regulated substrate recruitment to CRLs, including via substrate post-translational modification or various forms of regulation by small molecule plant hormones.
Cullin-RING ligases (CRLs) compose the largest class of E3 ubiquitin ligases. CRLs are modular, multisubunit enzymes, comprising interchangeable substrate receptors dedicated to particular Cullin-RING catalytic cores. Recent structural studies have revealed numerous ways in which CRL E3 ligase activities are controlled, including multimodal E3 ligase activation by covalent attachment of the ubiquitin-like protein NEDD8, inhibition of CRL assembly/activity by CAND1, and several mechanisms of regulated substrate recruitment. These features highlight the potential for CRL activities to be tuned in responses to diverse cellular cues, and for modulating CRL functions through small-molecule agonists or antagonists. As the second installment of a two-review series, this article focuses on recent structural studies advancing our knowledge of how CRL activities are regulated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by cleaving ubiquitin from specific protein substrates. How their activities are modulated in various cellular contexts remains ...poorly understood. The yeast deubiquitinase Ubp8 protein is recruited and activated by the SAGA complex and, together with Sgf11, Sus1, and Sgf73, forms a DUB module responsible for deubiquitinating histone H2B during gene expression. Here, we report the crystal structure of the complete SAGA DUB module, which features two functional lobes structurally coupled by Sgf73. In the “assembly lobe,” a long Sgf11 N-terminal helix is clamped onto the Ubp8 ZnF-UBP domain by Sus1. In the “catalytic lobe,” an Sgf11 C-terminal zinc-finger domain binds to the Ubp8 catalytic domain next to its active site. Our structural and functional analyses reveal a central role of Sgf11 and Sgf73 in activating Ubp8 for deubiquitinating histone H2B and demonstrate how a DUB can be allosterically regulated by its nonsubstrate partners.
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► Crystal structure of the SAGA histone H2B deubiquitinase module is determined ► Zinc-finger proteins Sgf11 and Sgf73 allosterically regulate the Ubp8 deubiquitinase ► The zinc-finger domain of Sgf11 helps shape the Ubp8 catalytic center ► The structure shows how nonsubstrate binding partners can activate a deubiquitinase
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present ...study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Supreme Court has recently insisted that plaintiffs must have standing for every claim that they raise. But this claim-specific approach to standing is at odds with established practice in ...several contexts, including rulings on the severability of statutes. Courts often permit plaintiffs to claim that statutory provisions should be invalidated pursuant to severability doctrine, without requiring that they have standing for those claims. This Article argues that existing practice for severability is a form of "supplemental standing." Supplemental standing is analogous to supplemental jurisdiction. It allows a plaintiff with standing for one claim to raise related claims, even if the plaintiff lacks standing for those other claims. Although the Supreme Court has purported to reject this concept, current law effectively grants supplemental standing for severability claims-and with good reason. When a court rules that part of a statute is unconstitutional, a ruling on severability is necessary to give effect to Congress's intent regarding the remainder of the statute. Supplemental standing permits those rulings, but claim-specific standing would often prevent them. In the severability context, therefore, supplemental standing is more faithful to the central purpose of standing doctrine: preserving the separation of powers. This Article contends that current practice also implicitly grants supplemental standing in additional contexts, including facial challenges to statutes and cases involving multiple plaintiffs. These examples suggest that, while the claim-specific approach to standing is a useful default rule, the Court has been wrong to insist on that approach as a categorical matter. The Article concludes by exploring additional circumstances in which shifting to a supplemental-standing approach appears to be justified on separation of powers grounds.
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CEKLJ, DOBA, IZUM, KILJ, NUK, PILJ, PNG, PRFLJ, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates ...Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous ...T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4(pos) T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4(pos) T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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