Evolution and Virulence Contributions of the Autotransporter Proteins YapJ and YapK of Yersinia pestis CO92 and Their Homologs in Y. pseudotuberculosis IP32953

ABSTRACT Yersinia pestis , the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis . Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis , while yapJ is unique to Y. pestis . All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ . Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis , any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models

Mechanisms of HIV-1 Nef Function and Intracellular Signaling

Advances in the last several years have enhanced mechanistic understanding of Nef induced CD4 and MHCI downregulation and have suggested a new paradigm for analyzing Nef function. In both of these cases, Nef acts by forming ternary complexes with significant contributions to stability imparted by non-canonical interactions. The mutational analyses and binding assays that have led to these conclusions are discussed. The recent progress has been dependent on conservative mutations and multi-protein binding assays. The poorly understood Nef functions of p21 activated protein kinase (PAK2) activation, enhancement of virion infectivity, and inhibition of immunoglobulin class switching are also likely to involve ternary complexes and non-canonical interactions. Hence, investigation of these latter Nef functions should benefit from a similar approach

Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism

Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body

Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis

A viral kinase mimics S6 kinase to enhance cell proliferation

Viruses usurp the host cell machinery to replicate, disseminate, and propagate themselves. Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a viral protein kinase (vPK) also known as ORF36. Using in silico modeling and biochemistry, we report that vPK/ORF36 displays limited homology to cellular S6 kinase B1 (S6KB1). Both kinases share overlapping substrates and can phosphorylate S6. However, unlike S6KB1, vPK augments S6 phosphorylation under conditions where mammalian target of rapamycin (mTOR) is inhibited. vPK modulates cellular proliferation and protein synthesis, augments anchorage independence, and enhances angiogenesis. Depletion of vPK/ORF36 during lytic replication inhibits the production of infectious virions, thereby underscoring the importance of this kinase during the KSHV life cycle. Our collective observations suggest that vPK may function as a constitutively active mimic of S6KB1

Evolution of a Signaling Nexus Constrained by Protein Interfaces and Conformational States

Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies

Active Nuclear Receptors Exhibit Highly Correlated AF-2 Domain Motions

Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 Å from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR–RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create “active-capable” receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators

Defining the Functional Domain of Programmed Cell Death 10 through Its Interactions with Phosphatidylinositol-3,4,5-Trisphosphate

Cerebral cavernous malformations (CCM) are vascular abnormalities of the central nervous system predisposing blood vessels to leakage, leading to hemorrhagic stroke. Three genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3) are involved in CCM development. PDCD10 binds specifically to PtdIns(3,4,5)P3 and OSM. Using threading analysis and multi-template modeling, we constructed a three-dimensional model of PDCD10. PDCD10 appears to be a six-helical-bundle protein formed by two heptad-repeat-hairpin structures (α1–3 and α4–6) sharing the closest 3D homology with the bacterial phosphate transporter, PhoU. We identified a stretch of five lysines forming an amphipathic helix, a potential PtdIns(3,4,5)P3 binding site, in the α5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Δ2KA), three (Δ3KA), and five (Δ5KA) K to A mutations. Δ2KA and Δ3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Δ5KA completely lacks all predicted binding residues. The WT, Δ2KA, and Δ3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Δ5KA abolishes binding to PtdIns(3,4,5)P3. Both Δ5KA and WT show similar secondary and tertiary structures; however, Δ5KA does not bind to OSM. When WT and Δ5KA are co-expressed with membrane-bound constitutively-active PI3 kinase (p110-CAAX), the majority of the WT is co-localized with p110-CAAX at the plasma membrane where PtdIns(3,4,5)P3 is presumably abundant. In contrast, the Δ5KA remains in the cytoplasm and is not present in the plasma membrane. Combining computational modeling and biological data, we propose that the CCM protein complex functions in the PI3K signaling pathway through the interaction between PDCD10 and PtdIns(3,4,5)P3

Trace elements in hemodialysis patients: a systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Hemodialysis patients are at risk for deficiency of essential trace elements and excess of toxic trace elements, both of which can affect health. We conducted a systematic review to summarize existing literature on trace element status in hemodialysis patients.</p> <p>Methods</p> <p>All studies which reported relevant data for chronic hemodialysis patients and a healthy control population were eligible, regardless of language or publication status. We included studies which measured at least one of the following elements in whole blood, serum, or plasma: antimony, arsenic, boron, cadmium, chromium, cobalt, copper, fluorine, iodine, lead, manganese, mercury, molybdenum, nickel, selenium, tellurium, thallium, vanadium, and zinc. We calculated differences between hemodialysis patients and controls using the differences in mean trace element level, divided by the pooled standard deviation.</p> <p>Results</p> <p>We identified 128 eligible studies. Available data suggested that levels of cadmium, chromium, copper, lead, and vanadium were higher and that levels of selenium, zinc and manganese were lower in hemodialysis patients, compared with controls. Pooled standard mean differences exceeded 0.8 standard deviation units (a large difference) higher than controls for cadmium, chromium, vanadium, and lower than controls for selenium, zinc, and manganese. No studies reported data on antimony, iodine, tellurium, and thallium concentrations.</p> <p>Conclusion</p> <p>Average blood levels of biologically important trace elements were substantially different in hemodialysis patients, compared with healthy controls. Since both deficiency and excess of trace elements are potentially harmful yet amenable to therapy, the hypothesis that trace element status influences the risk of adverse clinical outcomes is worthy of investigation.</p