USP5 enhances SGTA mediated protein quality control.

YesMislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance

Binding of SGTA to Rpn13 selectively modulates protein quality control

YesRpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation.BBSRC [grant number: BB/L006510/1] and the Wellcome Trust [grant number: 092107/Z/10/Z]. K.K. was supported by the UPStream network [EU, FP7, ITN project 290257

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region

Structure and function of the bacterial heterodimeric ABC transporter CydDC: stimulation of ATPase activity by thiol and heme compounds.

In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport

Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

YesThe ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation.This work was supported by grants from the BBSRC [H007202/1] and Wellcome Trust [097820/Z/11/A]

Expression, peroxisomal targeting and biochemical characterisation of a plant ABC protein, Comatose in S. cerevisiae

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Co-translational targeting and translocation of proteins to the endoplasmic reticulum

Co-translational protein targeting to the endoplasmic reticulum (ER), represents an evolutionary-conserved mechanism to target proteins into the secretory pathway. In this targeting pathway proteins possessing signal sequences are recognised at the ribosome by the signal recognition particle while they are still undergoing synthesis. This triggers their delivery to the ER protein translocation channel, where they are directly translocated into the ER. Here we review the current understanding of this translocation pathway and how molecular details obtained in the related bacterial system have provided insight into the mechanism of targeting and translocation. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum

Peroxisomal transport systems: roles in signaling and metabolism

Peroxisomes perform a range of different functions, including β-oxidation of fatty acids and synthesis and degradation of bioactive molecules. A notable feature of peroxisomes is their role in metabolic pathways which are shared between several subcellular compartments, including mitochondria, chloroplasts and cytosol. Transport across the peroxisomal membrane is therefore central to the co-ordination of metabolism. Although transport processes are required for import of substrates and cofactors, export of intermediates and products and the operation of redox shuttles, relatively few peroxisomal transporters have been identified to date. This chapter reviews the current evidence for and against different peroxisomal transport processes

The 2016 California policy to eliminate nonmedical vaccine exemptions and changes in vaccine coverage: An empirical policy analysis.

BackgroundVaccine hesitancy, the reluctance or refusal to receive vaccination, is a growing public health problem in the United States and globally. State policies that eliminate nonmedical ("personal belief") exemptions to childhood vaccination requirements are controversial, and their effectiveness to improve vaccination coverage remains unclear given limited rigorous policy analysis. In 2016, a California policy (Senate Bill 277) eliminated nonmedical exemptions from school entry requirements. The objective of this study was to estimate the association between California's 2016 policy and changes in vaccine coverage.Methods and findingsWe used a quasi-experimental state-level synthetic control analysis and a county-level difference-in-differences analysis to estimate the impact of the 2016 California policy on vaccination coverage and prevalence of exemptions to vaccine requirements (nonmedical and medical). We used publicly available state-level data from the US Centers for Disease Control and Prevention on coverage of measles, mumps, and rubella (MMR) vaccination, nonmedical exemption, and medical exemption in children entering kindergarten. We used county-level data individually requested from state departments of public health on overall vaccine coverage and exemptions. Based on data availability, we included state-level data for 45 states, including California, from 2011 to 2017 and county-level data for 17 states from 2010 to 2017. The prespecified primary study outcome was MMR vaccination in the state analysis and overall vaccine coverage in the county analysis. In the state-level synthetic control analysis, MMR coverage in California increased by 3.3% relative to its synthetic control in the postpolicy period (top 2 of 43 states evaluated in the placebo tests, top 5%), nonmedical exemptions decreased by 2.4% (top 2 of 43 states evaluated in the placebo tests, top 5%), and medical exemptions increased by 0.4% (top 1 of 44 states evaluated in the placebo tests, top 2%). In the county-level analysis, overall vaccination coverage increased by 4.3% (95% confidence interval [CI] 2.9%-5.8%, p ConclusionsIn this study, implementation of the California policy that eliminated nonmedical childhood vaccine exemptions was associated with an estimated increase in vaccination coverage and a reduction in nonmedical exemptions at state and county levels. The observed increase in medical exemptions was offset by the larger reduction in nonmedical exemptions. The largest increases in vaccine coverage were observed in the most "high-risk" counties, meaning those with the lowest prepolicy vaccine coverage. Our findings suggest that government policies removing nonmedical exemptions can be effective at increasing vaccination coverage