142 research outputs found

    Structural studies of the alpha-lactalbumin molten globule : implications for protein folding

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    Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1996.Vita.Includes bibliographical references.by Brenda Arlene Schulman.Ph.D

    Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

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    The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.</p

    The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

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    The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.Deutsche Forschungsgemeinschaft (DFG) http://dx.doi.org/10.13039/501100001659Max‐Planck‐Gesellschaft (MPG) http://dx.doi.org/10.13039/501100004189Peer Reviewe

    Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

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    Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity

    Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination

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    AbstractPrimary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli31-90) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3SPOP and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli31-90, the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli31-90 ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification

    Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

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    Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∌ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∌ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∌ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∌ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001

    Mutation in \u3ci\u3eATG5\u3c/i\u3e reduces autophagy and leads to ataxia with developmental delay

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    Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects’ cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health

    Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

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    Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades

    Lifetime history of indoor tanning in young people: a retrospective assessment of initiation, persistence, and correlates

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    <p>Abstract</p> <p>Background</p> <p>Despite educational and public health campaigns to convey the risks of indoor tanning, many individuals around the world continue to engage in this behavior. Few descriptive studies of indoor tanning have collected information pertaining to the lifetime history of indoor tanning, thereby limiting our ability to understand indoor tanning patterns and potentially target interventions for individuals who not only initiate, but continue to persistently engage in indoor tanning.</p> <p>Methods</p> <p>In-person interviews elicited detailed retrospective information on lifetime history of indoor tanning among white individuals (n = 401) under age 40 seen by a dermatologist for a minor benign skin condition. These individuals were controls in a case-control study of early-onset basal cell carcinoma. Outcomes of interest included ever indoor tanning in both males and females, as well as persistent indoor tanning in females - defined as females over age 31 who tanned indoors at least once in the last three or all four of four specified age periods (ages 11-15, 16-20, 21-30 and 31 or older). Multivariate logistic regression was used to identify sociodemographic and lifestyle correlates of ever and persistent indoor tanning in females.</p> <p>Results</p> <p>Approximately three-quarters (73.3%) of females and 38.3% of males ever tanned indoors, with a median age of initiation of 17.0 and 21.5, respectively. Among indoor tanners, 39.3% of females and 21.7% of males reported being burned while indoor tanning. Female ever indoor tanners were younger, had darker color eyes, and sunbathed more frequently than females who never tanned indoors. Using unique lifetime exposure data, 24.7% of female indoor tanners 31 and older persistently tanned indoors starting as teenagers. Female persistent indoor tanners drank significantly more alcohol, were less educated, had skin that tanned with prolonged sun exposure, and sunbathed outdoors more frequently than non-persistent tanners.</p> <p>Conclusions</p> <p>Indoor tanning was strikingly common in this population, especially among females. Persistent indoor tanners had other high-risk behaviors (alcohol, sunbathing), suggesting that multi-faceted behavioral interventions aimed at health promotion/disease prevention may be needed in this population.</p
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