Targeting Protein Quality Control Pathways in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a progressive neurodegenerative disease caused by a CAG/glutamine (polyQ) expansion in the androgen receptor (AR). Like many other age-dependent neurodegenerative diseases, SBMA is characterized by the buildup of misfolded proteins into nuclear aggregates and neurodegeneration. The mutant protein disrupts several cellular pathways, and decreasing levels of disease causing protein may circumvent several of the downstream pathological processes. Here we investigate the effects of manipulating protein quality control pathways in cell and animal models of SBMA, identifying novel therapeutic targets and advancing our understanding of molecular chaperones and their role in protein triage. Cells degrade proteins through two main pathways, autophagy and the ubiquitin proteasome pathway. Autophagy degrades cytosolic proteins in bulk, and increased autophagy has been shown to be beneficial in some models of protein aggregation diseases. Our results however, show that activating autophagy increases muscle wasting, while inhibiting autophagy significantly increases the lifespan and size of muscle fibers in a mouse model of SBMA. Our findings are surprising, and suggest that activation of autophagy in SBMA may exacerbate disease progression. The Hsp90/Hsp70-based chaperone machinery regulates the stabilization and degradation of Hsp90 clients through the proteasome, and presents an alternative therapeutic target to modulate proteostasis. Little is known however, about how this machinery functions to triage misfolded proteins, and few modulators of Hsp70 exist. Here we advance our understanding of chaperone machinery function, and present novel strategies to target Hsp70’s substrate affinity. We demonstrate that inhibiting Hsp70 function leads to accumulation of toxic AR, while increasing Hsp70 substrate affinity through overexpression of the co-chaperone Hip, or through treatment with a newly identified small molecule allosteric activator, promotes client protein ubiquitination and polyQ AR clearance. Both genetic and pharmacologic approaches to increase Hsp70 activity rescue disease phenotype in a Drosophila model of SBMA. Our results reveal a new therapeutic strategy of targeting Hsp70 to treat SBMA and perhaps other neurodegenerative diseases, while providing insights into the role of the chaperone machinery in protein quality control.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91413/1/amwang_1.pd

The X-Ray Position and Infrared Counterpart of the Eclipsing X-Ray Pulsar OAO 1657-415

We have measured the precise position of the 38-s eclipsing X-ray pulsar OAO 1657-415 with the Chandra X-Ray Observatory: RA = 17h00m48.90s, Dec = -41d39m21.6s, equninox J2000, error radius = 0.5 arcsec. Based on the previously measured pulsar mass function and X-ray eclipse duration, this 10.4-d high-mass X-ray binary is believed to contain a B supergiant companion. Deep optical imaging of the field did not detect any stars at the Chandra source position, setting a limit of V>23. However, near-IR imaging revealed a relatively bright star (J=14.1, H=11.9, K_s=10.7) coincident with the Chandra position, and we identify this star as the IR counterpart of OAO 1657-415. The IR colors and magnitudes and the optical non-detections for this star are all consistent with a highly reddened B supergiant (A_V= 20.4 +/- 1.3) at a distance of 6.4 +/- 1.5 kpc. This implies an X-ray luminosity of 3e36 erg/s (2-10 keV). IR spectroscopy can verify the spectral type of the companion and measure its radial velocity curve, yielding a neutron star mass measurement.Comment: 4 pages. ApJ in press (Vol. 573, July 10 issue

Isolation site influences virulence phenotype of serotype 14 Streptococcus pneumoniae strains belonging to multilocus sequence type 15

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains

A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease

Macroautophagy Is Regulated by the UPR–Mediator CHOP and Accentuates the Phenotype of SBMA Mice

Altered protein homeostasis underlies degenerative diseases triggered by misfolded proteins, including spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by a CAG/glutamine expansion in the androgen receptor. Here we show that the unfolded protein response (UPR), an ER protein quality control pathway, is induced in skeletal muscle from SBMA patients, AR113Q knock-in male mice, and surgically denervated wild-type mice. To probe the consequence of UPR induction, we deleted CHOP (C/EBP homologous protein), a transcription factor induced following ER stress. CHOP deficiency accentuated atrophy in both AR113Q and surgically denervated muscle through activation of macroautophagy, a lysosomal protein quality control pathway. Conversely, impaired autophagy due to Beclin-1 haploinsufficiency decreased muscle wasting and extended lifespan of AR113Q males, producing a significant and unexpected amelioration of the disease phenotype. Our findings highlight critical cross-talk between the UPR and macroautophagy, and they indicate that autophagy activation accentuates aspects of the SBMA phenotype

T Cell Detection of a B-Cell Tropic Virus Infection: Newly-Synthesised versus Mature Viral Proteins as Antigen Sources for CD4 and CD8 Epitope Display

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36–48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells

Genome-Wide Association Study Identifies Loci for Liver Enzyme Concentrations in Mexican Americans: The GUARDIAN Consortium.

ObjectivePopulations of Mexican American ancestry are at an increased risk for nonalcoholic fatty liver disease. The objective of this study was to determine whether loci in known and novel genes were associated with variation in aspartate aminotransferase (AST) (n = 3,644), alanine aminotransferase (ALT) (n = 3,595), and gamma-glutamyl transferase (GGT) (n = 1,577) levels by conducting the first genome-wide association study (GWAS) of liver enzymes, which commonly measure liver function, in individuals of Mexican American ancestry.MethodsLevels of AST, ALT, and GGT were determined by enzymatic colorimetric assays. A multi-cohort GWAS of individuals of Mexican American ancestry was performed. Single-nucleotide polymorphisms (SNP) were tested for association with liver outcomes by multivariable linear regression using an additive genetic model. Association analyses were conducted separately in each cohort, followed by a nonparametric meta-analysis.ResultsIn the PNPLA3 gene, rs4823173 (P = 3.44 × 10-10 ), rs2896019 (P = 7.29 × 10-9 ), and rs2281135 (P = 8.73 × 10-9 ) were significantly associated with AST levels. Although not genome-wide significant, these same SNPs were the top hits for ALT (P = 7.12 × 10-8 , P = 1.98 × 10-7 , and P = 1.81 × 10-7 , respectively). The strong correlation (r2  = 1.0) for these SNPs indicated a single hit in the PNPLA3 gene. No genome-wide significant associations were found for GGT.ConclusionsPNPLA3, a locus previously identified with ALT, AST, and nonalcoholic fatty liver disease in European and Japanese GWAS, is also associated with liver enzymes in populations of Mexican American ancestry

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids ​N-acetylneuraminic acid (​Neu5Ac) and ​N-glycolylneuraminic acid (​Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). ​Neu5Gc is synthesized from ​Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only ​Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize ​Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of ​Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains

The variable region of the pneumococcal pathogenicity island 1 is responsible for the unusually high virulence of a serotype 1 isolate

Streptococcus pneumoniae is the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region’s overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496�PPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496�PPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-�), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496�PPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing