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The AAA-ATPase (ATPases associated with diverse cellular activities) valosin-containing protein (VCP), is essential for many cellular pathways including but not limited to endoplasmic reticulum-associated degradation (ERAD), DNA damage responses, and cell cycle regulation. VCP primarily identifies ubiquitylated proteins in these pathways and mediates their unfolding and degradation by the 26S proteasome. This review summarizes recent research on VCP that has uncovered surprising new ways that this ATPase is regulated, new aspects of recognition of substrates and novel pathways and substrates that utilize its activity.

Endoplasmic reticulum (ER) protein homeostasis (proteostasis) is essential to facilitate proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis due to cell stress or mutations in ER proteins can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 significantly sensitizes cells to ER stress and activates canonical UPR signaling pathways. This in turn leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins belonging to ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we show that the p97 AAA-ATPase and its ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulate the prevalence of ER-mitochondria contacts. The p97-UBXD8 complex localizes to contacts and loss of this complex increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics of purified contacts demonstrates alterations in proteins regulating lipid metabolism upon loss of UBXD8. Furthermore, lipidomics studies indicate significant changes in distinct lipid species in UBXD8 knockout cells. We show that loss of p97-UBXD8 results in perturbed contacts due to an increase in membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts in p97-UBXD8 loss of function cells can be rescued by supplementation with unsaturated fatty acids or overexpression of SCD1. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark to a variety of human disorders such as neurodegeneration. Notably, we find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on p97-UBXD8.

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, mechanisms that regulate the deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from target proteins are poorly understood. We previously showed that the DUB USP-46 removes ubiquitin from the glutamate receptor GLR-1 and regulates it trafficking and degradation in C. elegans. We found that WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identify another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome promotes the abundance of USP-46, and this effect is non-additive with increased expression of WDR-48. We found that USP-46 is ubiquitinated, and expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the half-life of USP-46. A point mutant version of WDR-48 that disrupts binding to USP-46 is unable to promote USP-46 abundance in vivo. Together, these data support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism to control DUB availability and function.

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0–2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA–DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by β-galactosidase and β-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (= CGMCC 1.13590T = KCTC XXX).

The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, our results indicate that HIV-1 enhances its survivability in the host cell by utilizing this RNA methylation capability of DNMT2 to increase the stability of its own genome. Upon infection, DNMT2 re-localizes from the nucleus to the SGs and methylates HIV-1 RNA. This DNMT2-dependent methylation provided post-transcriptional stability to the HIV-1 RNA. Furthermore, DNMT2 overexpression increased the HIV-1 viral titre. This would suggest that HIV hijacks the RNA-processing machinery within the SGs to ensure its own survival in the host cell. Thus, our findings provide for a novel mechanism by which virus tries to modulate the host cell machinery to its own advantage.

A balance between protein synthesis and degradation is necessary to maintain cellular homeostasis. Failure to triage aberrant proteins may result in their accumulation and aggregation in the cytosol. The valosin-containing protein (VCP)-BCL2-associated athanogene 6 (BAG6) complex facilitates a wide variety of ubiquitin-mediated quality control events at the endoplasmic reticulum (ER), both prior to ER translocation and during ER-associated degradation (ERAD). However, how ubiquitylated clients associated with BAG6 are recognized by VCP for proteasomal degradation is presently unknown. We have identified UBXN1 as the VCP adaptor in BAG6-dependent processes occurring prior to ER insertion but not during ERAD. The loss of VCP-UBXN1 results in the inappropriate stabilization of ubiquitylated BAG6 clients and their accumulation in insoluble aggregates and sensitizes cells to proteotoxic stress. Our results identify how VCP is specifically targeted to ubiquitylated substrates in the BAG6 triage pathway and suggest that the degradation of ubiquitylated clients by the proteasome is reliant on the association of UBXN1 with ubiquitylated substrates and the catalytic activity of VCP.

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mono-infected and HIV-M. bovis BCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.

The draft genome of Thermocrinis jamiesonii GBS1 T is 1,315,625 bp in 10 contigs and encodes 1,463 predicted genes. The presence of sox genes and various glycoside hydrolases and the absence of uptake NiFe hydrogenases ( hyaB ) are consistent with a requirement for thiosulfate and suggest the ability to use carbohydrate polymers.

Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination.

A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.

Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

Mycobacterium tuberculosis (Mtb) is the most common co-infection in HIV patients and a serious co-epidemic. Apart from increasing the risk of reactivation of latent tuberculosis (TB), HIV infection also permits opportunistic infection of environmental non-pathogenic mycobacteria. To gain insights into mycobacterial survival inside host macrophages and identify mycobacterial proteins or processes that influence HIV propagation during co-infection, we employed proteomics approach to identify differentially expressed intracellular mycobacterial proteins during mono- and HIV co-infection of human THP-1 derived macrophage cell lines. Of the 92 proteins identified, 30 proteins were upregulated during mycobacterial mono-infection and 40 proteins during HIV-mycobacteria co-infection. We observed down-regulation of toxin-antitoxin (TA) modules, up-regulation of cation transporters, Type VII (Esx) secretion systems, proteins involved in cell wall lipid or protein metabolism, glyoxalate pathway and branched chain amino-acid synthesis during co-infection. The bearings of these mycobacterial factors or processes on HIV propagation during co-infection, as inferred from the proteomics data, were validated using deletion mutants of mycobacteria. The analyses revealed mycobacterial factors that possibly via modulating the host environment, increased viral titers during co-infection. The study provides new leads for investigations towards hitherto unknown molecular mechanisms explaining HIV-mycobacteria synergism, helping address diagnostics and treatment challenges for effective co-epidemic management.