Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase

Thr(93), Ser(94), Thr(140), and Ser(306) are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild-type enzyme, indicating there is no appreciable change in secondary structure. T93A exhibits 0.5% of the V(max) of wild-type ASL with a 10-fold increase in K(m) for adenylosuccinate. S94A has 65% of the V(max) of wild-type ASL with little change in K(m). T140A exhibits 0.03% of the activity of wild-type enzyme with an 11-fold increase in K(m). S306A has 0.4% of the V(max) of wild-type ASL with a sevenfold increase in K(m). Measurements of the pH-V(max) profile reveal a pK(2) value for S94A of 7.83 and S306A of 7.65, in contrast to 8.24 for the wild-type enzyme and 8.42 for T93A. Thr(93) may orient adenylosuccinate optimally for catalysis, while Ser(94) stabilizes protonated His(89), a determinant of pK(2). Thr(140) may, through hydrogen bonding, interact with Asn(270), an amino acid essential for catalysis. Ser(306) may be involved in a hydrogen bond network that ultimately stabilizes protonated His(68), which is probably the general acid in the reaction of enzyme with substrate. The results of this paper demonstrate the importance in the catalytic function of ASL of hydrogen bonds and hydrogen bonding networks involving serine and threonine.     

Species barriers for chronic wasting disease by in vitro conversion of prion protein

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP^CWD of elk can readily induce the conversion of normal cervid PrP (PrP^C) molecules to a protease-resistant form, but is less efficient in converting the PrP^C of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP^CWD-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrP^C from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP^C can apparently overcome the structural barriers between more distant species.     

Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues

The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues. The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy. The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA. Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.     

Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.

Methodology/Principal Findings

Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.

Conclusion/Significance

We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.     

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

Background  

Insensitivity of advanced-stage prostate cancer to androgen ablation therapy is a serious problem in clinical practice because it is associated with aggressive progression and poor prognosis. Targeted therapeutic drug discovery efforts are thwarted by lack of adequate knowledge of gene(s) associated with prostate tumorigenesis. Therefore there is the need for studies to provide leads to targeted intervention measures. Here we propose that stable expression of U94, a tumor suppressor gene encoded by human herpesvirus 6A (HHV-6A), could alter gene expression and thereby inhibit the tumorigenicity of PC3 cell line. Microarray gene expression profiling on U94 recombinant PC3 cell line could reveal genes that would elucidate prostate cancer biology, and hopefully identify potential therapeutic targets.     

Involvement of Aminopeptidase N (CD13) in Infection of Human Neural Cells by Human Coronavirus 229E

Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800–806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.     

Cellular isoform of the scrapie agent protein participates in lymphocyte activation

The scrapie agent protein (Sp33-37 or PrPSc) is the disease-associated isoform of a normal cellular membrane protein (Cp33-37 or PrPC) of unknown function. We report that normal human lymphocytes and lymphoid cell lines, but not erythrocytes or granulocytes, express PrPC mRNA and protein. PrPC is detectable on the surface of lymphocytes; the surface immunoreactivity is sensitive to phosphatidylinositol-specific phospholipase C, indicating glycosyl-phosphatidylinositol membrane anchorage. Lymphocyte PrPC surface abundance is increased by cell activation, and polyclonal antibodies to PrPC suppress mitogen-induced activation. We conclude that PrPC is a lymphocyte surface molecule that may participate in cell activation.     

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

Background  

Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone.