Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Darby, John F.; Krysztofinska, E.; Simpson, Peter; Simon, Aline C.; Leznicki, Pawel; Sriskandarajah, Newran; Bishop, David S.; Hale, Lisa R.; Alfano, Caterina; Conte, Maria R.; Martínez-Lumbreras, Santiago; Thapaliya, Arjun; High, Stephen; Isaacson, Rivka L.

doi:10.1371/journal.pone.0113281

Cited by 21 publications

(41 citation statements)

References 38 publications

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“…Because SGTA impacts the virus-Hsc70 interaction, we sought to determine whether SGTA's ability to associate with Hsc70 via SGTA's central TPR domain (Fig. 4A) (32-34), as well as SGTA's N-terminal domain, which mediates homodimerization and the recruitment cellular adapters, including the Ubl4A-Tric35-Bag6 complex (33)(34)(35)(36), is important during ER extraction and cytosol arrival of the virus. Since SGTA's C-terminal domain is limited to substrate binding (31,37,38) and unlikely to control Hsc70's function, we did not characterize this domain in this study.…”

Section: Resultsmentioning

confidence: 99%

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

Dupzyk

Williams

Bagchi

et al. 2017

J Virol

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Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.IMPORTANCE How a nonenveloped virus transports across a biological membrane to cause infection remains mysterious. One enigmatic step is whether host cytosolic components are co-opted to transport the viral particle into the cytosol. During ERto-cytosol membrane transport of the nonenveloped polyomavirus SV40, a decisive infection step, a cytosolic complex composed of Hsc70-SGTA-Hsp105 was previously shown to associate with the ER membrane. SGTA and Hsp105 have been shown to extract SV40 from the ER and transport the virus into the cytosol. We demonstrate here a critical role of Hsc70 in SV40 ER-to-cytosol penetration and reveal how SGTA controls Hsc70 to impact this process.KEYWORDS chaperones, endoplasmic reticulum, membrane transport, simian virus 40 T he molecular basis by which nonenveloped viruses penetrate biological membranes to gain entry into the host cytosol and cause infection remains mysterious (1). For enveloped viruses, fusion between the viral and a host membrane enables the core viral particle to reach the cytosol (2). However, since nonenveloped viruses lack a surrounding lipid bilayer, their membrane transport mechanisms are likely distinct from those of enveloped viruses. Although the precise molecular mechanism remains unclear, a general principle describing nonenveloped membrane translocation has nonetheless emerged. Upon trafficking to the site of membrane penetration, host cues, including cellular proteases, chaperones, reductases, and low pH, act on the viral particle to impart conformational changes. As a consequence, the conformationally altered virus becomes hydrophobic, enabling it to engage and disrupt the limiting membrane. Alternatively, the structural change may release a lytic peptide previously hidden in the native virus, which in turn integrates into and impairs the integrity of the membrane. Regardless of the mechanism, these reactions prime the viral (or subviral)

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Section: Resultsmentioning

confidence: 99%

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

Dupzyk

Williams

Bagchi

et al. 2017

J Virol

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“…Microscale thermophoresis protein-protein interaction studies were performed on the Monolith NT.115 (NanoTemper Technologies, Munich, Germany) using fluorescently labeled proteins as previously described [36]. Purified PDGF-BB was labeled using the Monolith NT protein labeling kit RED-NHS (Amine Reactive) dye (NanoTemper Technologies GmbH).…”

Section: Methodsmentioning

confidence: 99%

PDGF-BB/KLF4/VEGF Signaling Axis in Pulmonary Artery Endothelial Cell Angiogenesis

Liang

Yu²,

Chen³

et al. 2017

Cell Physiol Biochem

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Background: Accumulating evidence suggests that platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor(VEGF) play a role in the progression of pulmonary arterial hypertension (PAH).Since chronic hypoxia is responsible for intimal hyperplasia and disordered angiogenesis of pulmonary arteries, which are histological hallmarks of PAH, we explored the role of the PDGF-BB/KLF4/VEGF signaling axis in the angiogenesis of pulmonary artery endothelial cells (PAECs). Methods: Adult male Wistar rats were used to study hypoxia-induced or monocrotaline (MCT)-induced right ventricular (RV) remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and RV vessels were performed. Results: The results revealed that both the PDGF receptor-tyrosine kinase inhibitor imatinib and the multi-targeted VEGF and PDGF receptor inhibit or sunitinib malate reversed hypoxia-induced increases in right ventricular systolic pressure (RVSP), right ventricular function and thickening of the medial walls. Mechanistically VEGF/VEGFR and PDGF/PDGFR formed a biological complex. We also showed that PDGF-BBincreasedKLF4 promoter activity transcriptionally activating VEGF expression, which regulates PAEC proliferation; migration; and the cell-cycle transition from G0/G1phase to S phase and G2/M-phase and eventually leads to PAEC angiogenesis Conclusion: Our study indicates that hypoxia-induced angiogenesis of PAECs is associated with increased levels of PDGF-BB/KLF4/VEGF, which contribute to pulmonary vascular remodeling. Overall, our study contributes to a better understanding of PAH pathogenesis.

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“…ITC experiments were performed using an ITC-200 microcalorimeter from Microcal (GE Healthcare) at 25 °C following the standard protocol as reported previously7. Proteins or peptide samples were prepared in 10 mM potassium phosphate pH 6.0, 100 mM NaCl, 250 μM TCEP.…”

Section: Methodsmentioning

confidence: 99%

“…SGTA and the BAG6 complex were initially discovered through their role in the biogenesis of tail-anchored membrane proteins and subsequently shown to regulate the ubiquitination and proteasomal degradation of MLPs23. The role of SGTA and BAG6 in both processes relies on multiple transient, and subtly discriminated, interactions with diverse binding partners and effectors45678910. These interactions contribute to a proposed BAG6/SGTA quality control cycle that can direct hydrophobic substrates towards either ubiquitination and proteasomal degradation (MLPs) or membrane insertion (tail-anchored proteins)2345111213.…”

mentioning

confidence: 99%

“…The BAG6 complex comprises at least two copies each of BAG6, UBL4A (ubiquitin-like protein 4A) and TRC35 (transmembrane recognition complex 35) and displays two different kinds of UBL domains; UBL4A_UBL and BAG6_UBL111415. SGTA, on the other hand, is a homodimer, tightly constrained at its N-terminal domain71617. It has a central TPR domain capable of direct interaction with both Hsp70 and Hsp90 chaperones, proteasomal subunits and a variety of disease-related proteins618, and a C-terminal domain capable of binding hydrophobic substrates19.…”

mentioning

confidence: 99%

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SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

Thapaliya

Nyathi

Martínez-Lumbreras

et al. 2016

Sci Rep

Self Cite

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The fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context.

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Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Cited by 21 publications

References 38 publications

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

PDGF-BB/KLF4/VEGF Signaling Axis in Pulmonary Artery Endothelial Cell Angiogenesis

SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

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