Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.     

Homoplasy in genome-wide analysis of rare amino acid replacements: the molecular-evolutionary basis for Vavilov's law of homologous series

Background  

Rare genomic changes (RGCs) that are thought to comprise derived shared characters of individual clades are becoming an increasingly important class of markers in genome-wide phylogenetic studies. Recently, we proposed a new type of RGCs designated RGC_CAMs (after Conserved Amino acids-Multiple substitutions) that were inferred using genome-wide identification of amino acid replacements that were: i) located in unambiguously aligned regions of orthologous genes, ii) shared by two or more taxa in positions that contain a different, conserved amino acid in a much broader range of taxa, and iii) require two or three nucleotide substitutions. When applied to animal phylogeny, the RGC_CAM approach supported the coelomate clade that unites deuterostomes with arthropods as opposed to the ecdysozoan (molting animals) clade. However, a non-negligible level of homoplasy was detected.     

The mechanobiology theory of the development of medical device-related pressure ulcers revealed through a cell-scale computational modeling framework

Biomechanics and Modeling in Mechanobiology - Pressure ulcers are localized sites of tissue damage which form due to the continuous exposure of skin and underlying soft tissues to sustained...     

Colon stem cell and crypt dynamics exposed by cell lineage reconstruction

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems.     

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and β generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC β inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC β as another PKC β inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC β, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.     

Analysis of rare amino acid replacements supports the Coelomata clade

The recent analysis of a novel class of rare genomic changes, RGC_CAMs (after conserved amino acids-multiple substitutions), supported the Coelomata clade of animals as opposed to the Ecdysozoa clade (Rogozin et al. 2007). A subsequent reanalysis, with the sequences from the sea anemone Nematostella vectensis included in the set of outgroup species, suggested that this result was an artifact caused by reverse amino replacements and claimed support for Ecdysozoa (Irimia et al. 2007). We show that the internal branch connecting the sea anemone to the bilaterian animals is extremely short, resulting in a weak statistical support for the Coelomata clade. Direct estimation of the level of homoplasy, combined with taxon sampling with different sets of outgroup species, reinforces the support for Coelomata, whereas the effect of reversals is shown to be relatively minor.     

Nonmuscle Myosin Heavy Chain IIA Is a Critical Factor Contributing to the Efficiency of Early Infection of Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.