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During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62 mice, global p62 and Ucp1-Cre p62 mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.
The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1.
© 2019 Zhao et al.
Clostridioides (formerly Clostridium) difficile is a Gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in US hospitals. The disease state is usually preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe diarrhoea. C. difficile infection is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB) and the C. difficile transferase toxin (CDT). Whereas TcdA and TcdB are considered the primary virulence factors, recent studies suggest that CDT increases the severity of C. difficile infection in some of the most problematic clinical strains. To better understand how CDT functions, we used cryo-electron microscopy to define the structure of CDTb, the cell-binding component of CDT. We obtained structures of several oligomeric forms that highlight the conformational changes that enable conversion from a prepore to a β-barrel pore. The structural analysis also reveals a glycan-binding domain and residues involved in binding the host-cell receptor, lipolysis-stimulated lipoprotein receptor. Together, these results provide a framework to understand how CDT functions at the host cell interface.
The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
Copyright © 2019 Elsevier Ltd. All rights reserved.
This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M PAM activity and improved clearance and protein binding profiles.
Copyright © 2019 Elsevier Ltd. All rights reserved.
The voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor-pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.
The five articles in this multi-author review in CMLS provide examples of multi-functionality of proteins belonging to several families. Distinct structural features of proteins suggesting multi-functionality are emphasized: intrinsically disordered elements that can "mold" themselves to fit various binding partners, as well as short linear motifs within larger proteins that perform particular functions. Although only a few protein families are discussed in detail, the conclusions apply to numerous, if not all, proteins. Multi-functionality of virtually every protein implies that the manipulation of its expression levels by over-expression, knockdown, or knockout affects every one of its functions, known and unknown, so that the results of these experiments must be interpreted with this complexity in mind. Particular functions in a multi-functional protein are often fulfilled by identifiable smaller elements that can be expressed separately. Identification of mono-functional elements of a multi-functional protein paves the way to the construction of novel precisely targeted molecular tools for selective manipulation of cellular signaling that can be used for mechanistic studies in cell biology, as well as for therapeutic purposes.
Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP K 170 nM, SPR K 460 nM) and cell activity (IC 38 µM). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.
Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis. While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.
Copyright © 2019 American Society for Microbiology.