Human cytomegalovirus pUL29/28 and pUL38 repression of p53-regulated p21CIP1 and caspase 1 promoters during infection

During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs

Human Cytomegalovirus UL28 and UL29 Open Reading Frames Encode a Spliced mRNA and Stimulate Accumulation of Immediate-Early RNAs▿

We have identified a spliced transcript that contains sequences from the HCMV UL29 and UL28 open reading frames. It contains amino-terminal UL29 sequences followed by UL28 sequences, and it includes a poly(A) signal derived from the 3′-untranslated region following the UL26 open reading frame. UL29/28 RNA is expressed with early kinetics, and a virus containing a FLAG epitope inserted at the amino terminus of UL29 expressed a tagged ∼79-kDa protein, pUL29/28, that was detected at 6 h postinfection. The virus also expressed a less-abundant tagged 41-kDa protein, which corresponds in size to a protein that could be produced by translation of an unspliced UL29/28 transcript. Consistent with this prediction, both unspliced and spliced UL29/28 transcript was present in RNA isolated from polysomes. FLAG-tagged protein from the UL29/28 locus accumulated within nuclear viral replication centers during the early phase of infection. Late after infection it was present in the cytoplasm as well, and the protein was present and resistant to proteinase treatment in partially purified preparations of viral particles. Disruption of the UL29/28 locus by mutation resulted in a 10-fold decrease in the levels of DNA replication along with a similar reduction in virus yield. Quantitative reverse transcription-PCR analysis revealed an ∼2-fold decrease in immediate-early gene expression at 4 to 10 h postinfection compared to the wild-type virus, and transient expression of pUL29/28 activated the major immediate-early promoter. Our results argue that the UL29/28 locus contributes to activation of immediate-early gene expression

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress

Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection

Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2,905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.The following grants are acknowledged: National Institutes of Health P41 GM108569, R01 GM067193 to NLK, P01 AI112522 to NLK and MMA, and U19 A1063603 to DRS

Applying Label-Free Quantitation to Top Down Proteomics

With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is ∼100–400 μg of total protein for each biological replicate, and the outputs are graphical displays depicting statistical confidence metrics for each proteoform (<i>i.e</i>., a volcano plot and representations of the technical and biological variation). A key part of the pipeline is the hierarchical linear model that is tailored to the original design of the study. Here, we apply this new pipeline to measure the proteoform-level effects of deleting a histone deacetylase (<i>rpd3</i>) in S. cerevisiae. Over 100 proteoform changes were detected above a 5% false positive threshold in WT vs the Δ<i>rpd3</i> mutant, including the validating observation of hyperacetylation of histone H4 and both H2B isoforms. Ultimately, this approach to label-free top down proteomics in discovery mode is a critical technical advance for testing the hypothesis that whole proteoforms can link more tightly to complex phenotypes in cell and disease biology than do peptides created in shotgun proteomics

Applying Label-Free Quantitation to Top Down Proteomics

With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is ∼100–400 μg of total protein for each biological replicate, and the outputs are graphical displays depicting statistical confidence metrics for each proteoform (<i>i.e</i>., a volcano plot and representations of the technical and biological variation). A key part of the pipeline is the hierarchical linear model that is tailored to the original design of the study. Here, we apply this new pipeline to measure the proteoform-level effects of deleting a histone deacetylase (<i>rpd3</i>) in S. cerevisiae. Over 100 proteoform changes were detected above a 5% false positive threshold in WT vs the Δ<i>rpd3</i> mutant, including the validating observation of hyperacetylation of histone H4 and both H2B isoforms. Ultimately, this approach to label-free top down proteomics in discovery mode is a critical technical advance for testing the hypothesis that whole proteoforms can link more tightly to complex phenotypes in cell and disease biology than do peptides created in shotgun proteomics