Adam Oberstein

Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein.

Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-XL. However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-XL. We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-XL at 2.5Å resolution. Reminiscent of previously determined Bcl-XL-BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-XL. These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.

Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry.

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix α4 approximately 10 Å below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix α5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix α5.

Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus‐coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC‐labeled human fibroblasts during infection with either wild‐type HCMV or a pUL97 kinase‐dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high‐resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

Cellular responses to human cytomegalovirus infection: Induction of a mesenchymal-to-epithelial transition (MET) phenotype

Significance Human cytomegalovirus (HCMV) causes birth defects and serious disease in immunocompromised patients. We do not fully understand the cellular processes that HCMV manipulates during infection. A holistic understanding of the cellular response to HCMV will help clarify mechanisms that underlie its replication and spread. This work uses systems virology to globally map the host response to HCMV infection. Our method identifies unappreciated pathways modulated by HCMV, including mesenchymal-to-epithelial transition (MET), an important developmental pathway involved in epithelial tissue formation, wound healing, and cancer metastasis. Our findings demonstrate that HCMV induces MET and raise the possibility that the transition influences not only viral pathogenesis but also the behavior of normal and diseased cells within an infected human host. Human cytomegalovirus (HCMV) is the prototypical human β-herpes virus. Here we perform a systems analysis of the HCMV host-cell transcriptome, using gene set enrichment analysis (GSEA) as an engine to globally map the host–pathogen interaction across two cell types. Our analysis identified several previously unknown signatures of infection, such as induction of potassium channels and amino acid transporters, derepression of genes marked with histone H3 lysine 27 trimethylation (H3K27me3), and inhibition of genes related to epithelial-to-mesenchymal transition (EMT). The repression of EMT genes was dependent on early viral gene expression and correlated with induction E-cadherin (CDH1) and mesenchymal-to-epithelial transition (MET) genes. Infection of transformed breast carcinoma and glioma stem cells similarly inhibited EMT and induced MET, arguing that HCMV induces an epithelium-like cellular environment during infection.

Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

Significance Human CMV (HCMV) is a major cause of birth defects, an opportunistic infection in untreated HIV/AIDS, and a life-threatening complication in immunosuppressed transplant patients. HCMV infects multiple different cell types, so understanding mechanisms supporting the entry of the virus into different cells is crucial to deciphering its pathogenesis. Two viral glycoprotein complexes (trimeric and pentameric complexes) are known to control HCMV tropism in different cell types. Here we have refined our understanding of the mechanism by which the cell-surface receptor tyrosine kinase, PDGF receptor-α, supports the entry of HCMV into fibroblasts. Human CMV (HCMV) exhibits a broad cell tropism that depends on two virion glycoprotein complexes: a trimeric complex (gH/gL/gO) that facilitates viral infection primarily in fibroblasts and a pentameric complex (gH/gL/pUL128-pUL130-pUL131A) that mediates infection in epithelial and endothelial cells. We performed genome-wide CRISPR screens in which the PDGF receptor-α (PDGFRα) was identified as the most significant cellular gene product essential for infection by HCMV virions containing only trimeric complex (trimer-only virus). Trimer-only virus did not enter PDGFRα knockout fibroblasts. By using knockout fibroblasts, the extracellular domain of PDGFRα required for virus entry was mapped, and the intracellular tyrosine kinase domain was shown to be nonessential. In addition, direct cell-to-cell spread of virus from knockout cells transfected with trimer-only viral DNA was blocked, despite the production of infectious virus in the transfected cells. In contrast to trimer-only virus, wild-type HCMV virions containing both trimeric and pentameric complexes entered PDGFRα knockout cells, reinforcing the view that fibroblasts contain a second, independent receptor for the pentameric complex. Importantly, however, wild-type virus entered the knockout fibroblasts at reduced efficiency compared with parental fibroblasts, arguing that the cellular receptor for the virion pentameric complex is limiting or that virions are produced containing different relative amounts of the two glycoprotein complexes. Finally, ectopic expression of PDGFRα in ARPE-19 epithelial cells and THP-1 monocytic cells, which have little to no endogenous PDGFRα expression, markedly enhanced their susceptibility to trimer-only virions. In sum, our data clarify several key determinants of HCMV tropism.