Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.16.379206v1?rss=1

Authors: Takahashi, M., Seino, R., Ezoe, T., Ishiyama, M., Ueno, Y.

Abstract:

The plasma membrane (PM) plays a critical role in many cellular processes, and PM dysfunction is a key biomarker related to the cell status and several diseases. Imaging techniques using small fluorescent probes have become increasingly important tools for visualizing living cells, and particularly, their PMs. Among the commercially available PM-specific probes, PKH dyes are widely used; however, the utility of these dyes is limited by their short membrane retention times and high cytotoxicity. Herein, PlasMem Bright Green and Red are implemented as new PM-specific fluorescent probes, which employ a polycyclic aromatic fluorophore to improve their retention ability and a strong acid moiety to reduce their transmembrane diffusion and cytotoxicity. We demonstrate that the long retention and low cytotoxicity of the PlasMem Bright dyes enable them to be applied for observing neuronal PMs and monitoring PM dynamics involving the endocytic pathway. Furthermore, we successfully detected mitochondria in nerve axons over long periods of time using PlasMem Bright dyes. Finally, the combined use of exosome staining probes and PlasMem Bright dyes allowed clear visualization of the endocytic pathway.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.16.385187v1?rss=1

Authors: Oganesyan, I., Lento, C., Tandon, A., Wilson, D. J.

Abstract:

Both normal and pathological functions of -synuclein (SN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of SN upon membrane complexation will clarify molecular mechanisms associated with SN-linked pathologies, including Parkinsons disease (PD), Multiple Systems Atrophy and other synucleinopathies. In this work, Time-Resolved ElectroSpray Ionization Hydrogen/ Deuterium Exchange Mass Spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of SNs conformational transitions as it undergoes complexation with nanodisc membrane mimics. Using this approach, SN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have a little impact on the SN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the SN conformational ensemble. Ultimately, we identify a region corresponding residues 19-28 and 45-57 of the SN sequence that is uniquely impacted by interactions with amyloidogenic lipid membranes and may therefore play a critical role in pathogenic aggregation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.16.384339v1?rss=1

Authors: Ngah, Y. E., Cho, F. N., Etagha, B. S., Fusi, N. G., Francisca, N., Ikomey, M. G., Ibrahim, N.

Abstract:

Introduction The incidence of hepatotoxicity is life-threatening and can result to an end-stage liver disease in long-term patients on combined antiretroviral therapy (cART). Our study sought to evaluate the incidence and predictors of cART-induced hepatotoxicity (CIH) among long term users on cART in a rural District hospital. Methods This was a hospital-based cross-sectional study in the Bali District Hospital. Spectrophotometric method was use for the quantitative measurement of alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels. Patients with elevations of both ALT and AST were considered CIH. The Chi ({chi} 2 ) square test, ANOVA and Kaplan Meier log-ranked/ survival analyses were used to analyse the data. Results Of the 350 participants enrolled [156 (44.6%) males and 194 (55.4%) females], aged 43.87 {+/-} 0.79 years (range 20 - 84 years) included in this analysis, 26 (4.4%) experienced moderate CIH. We observed 57 (16.3%), 62 (17.7%) and 238 (68%) elevated levels ALT + AST, ALT and AST respectively. Two independent predictive factors of CIH were, the male sex and alcoholism during the study period. Conclusion The prevalence of CIH in HIV-infected patients in Bali was lower than that observed in previous studies. The duration of therapy had no influence on the frequency of CIH. Alcoholism and smoking showed significant differences in the development of CIH.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.16.384594v1?rss=1

Authors: Brun, J., Vasiljevic, S., Gangadharan, B., Hensen, M., Chandran, A. V., Hill, M. L., Kiappes, J. L., Dwek, R. A., Alonzi, D. S., Struwe, W. B., Zitzmann, N.

Abstract:

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their cellular secretion pathways are unique, resulting in different protein glycosylation and secretion, which may have implications for the resulting immune response and future vaccine design.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.15.383679v1?rss=1

Authors: Knight, K. M., Ghosh, S., Campbell, S. L., Lefevre, T. J., Olsen, R. H. J., Smrcka, A. V., Valentin, N. H., Yin, G., Vaidehi, N., Dohlman, H. G.

Abstract:

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.14.363911v1?rss=1

Authors: Li, J., Huang, Q.

Abstract:

Oat protein isolate (OPI) is among the plant proteins with valuable functionalities (e.g. emulsification) during daily supplement intake. Understanding their structures helps to manipulate oat proteins at small scale, which enables the appropriate deployment of their functions. Based upon such understanding, the molecular structure of oat protein isolate (OPI) in aqueous medium was investigated by synchrotron small angle X ray scattering (SAXS), and this study allows a structural reconstitution of OPI in aqueous medium. Besides, this SAXS study is complimentary to the previous study (Liu et al. J. Agric. Food Chem. 2009, 57, 4552 to 4558). From form factor fitting, we confirmed that OPI aqueous solutions at low concentrations (0.3~2 mg/mL) obtained a disk conformation (41.4*41.4*10.2 A3). Once protein concentration increased to 5 mg/mL and 10 mg/mL, the individual disk proteins formed large dimensional rod like aggregates, which was evidenced by the analyses of effective structure factor and pair distribution function (PDF). Based on the PDF results, the ab initio models of OPI particles at low concentrations (0.3 mg/mL to 2.0 mg/mL) were restored by using GASBOR algorithm. Finally, we found that weak attraction between OPI particles occurred, which was verified by second virial coefficient and pair potential.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.14.381277v1?rss=1

Authors: Johansson, E., Diffley, J. F. X.

Abstract:

Single-stranded DNA breaks, including simple nicks, are amongst the most common forms of DNA damage in cells. They can be readily repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a 'collapsed' replication fork in which the nick is converted into a single-ended double-strand DNA break. Attention has largely focused on the processes by which this broken end is used to prime replication restart. We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a second fork converging on the collapsed fork offers additional opportunities for repair, but also generates a substrate that can promote localized re-replication. We have modelled this with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.13.381293v1?rss=1

Authors: Nguyen, G. T. T., Sutinen, A., Raasakka, A., Muruganandam, G., Loris, R., Kursula, P.

Abstract:

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive towards classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point towards allosteric mechanisms in regulating GDAP1 oligomeric state and function.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.13.381962v1?rss=1

Authors: Cheng, C. L., Wong, M. K., Li, Y., Hochstrasser, M.

Abstract:

The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.11.13.381988v1?rss=1

Authors: Saumya, K. U., Gadhave, K., Kumar, A., Giri, R.

Abstract:

Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During ZIKV polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Since misfolding and aggregation propensity of transmembrane proteins are now increasingly recognized and has been linked to several proteopathic disorders. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed aggregation behavior of CA peptide using dye-binding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM and AFM) revealed characteristic amyloid-like fibrils. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection.

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