Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2–5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis

PROTEOLYTIC MATURATION AND PROTEIN DEGRADATION IN ARABIDOPSIS THALIANA CHLOROPLASTS

Proteolysis is crucial for the maturation, regulation and recycling of the chloroplast proteome. Although several dozen chloroplast proteases are known, information concerning their substrates and functions is limited. In particular, little is known about the structural features of substrates that trigger their proteolysis. Most chloroplast proteins are nuclear encoded and are targeted through an N-terminal chloroplast transit peptide (cTP) that is removed by stromal processing peptidase (SPP). To better understand proteolytic maturation, the soluble N-terminal proteome of the Arabidopsis thaliana chloroplast was characterized. A cTP cleavage motif was observed that suggests other peptidases, in addition to SPP, are involved in chloroplast protein maturation. There was a clear preference for small uncharged amino acids at the processed protein N-terminus suggesting the existence of a chloroplast specific ‘N-end rule’. The soluble chloroplast peptidases PREP and OOP have been shown to degrade small polypeptides in vitro and are thought to be responsible for removal of cTP fragments and other degradation products. The CLP protease system can degrade intact protein substrates with the aid of ATP dependent (AAA+) CLPC chaperones that unfold and feed substrates into the CLP proteolytic core. An array of proteomic tools were used to compare Arabidopsis mutants deficient in the above peptidases with wild type. Degradation products, including cTPs, were found to accumulate in peptidase mutants indicative, of rate-limited or blocked degradation pathways. Incomplete or altered N-terminal maturation for chloroplast proteins was dependent on the type and severity of the peptidase deficiency. These results provide molecular details to help explain dwarf, chlorotic mutant phenotypes and demonstrate the interplay between protein import, proteolytic processing and the downstream degradation of damaged or unwanted proteins in the chloroplast. Substrate and sequence cleavage specificity was determined for soluble chloroplast glutamyl-endopeptidase (CGEP) and the plastoglobule localized metallopeptidase PGM48. Structural models were used to predict peptidase substrate binding mechanisms

Immune Response and Alveolar Bone Resorption in a Mouse Model of Treponema denticola Infection▿

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host's immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls

Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis

Abstract Almost all cells require thiamin, vitamin B1 (B1), which is synthesized via the coupling of thiazole and pyrimidine precursors. Here we demonstrate that 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cHET) is a useful in vivo B1 precursor for representatives of ubiquitous marine picoeukaryotic phytoplankton and Escherichia coli – drawing attention to cHET as a valuable exogenous micronutrient for microorganisms with ecological, industrial, and biomedical value. Comparative utilization experiments with the terrestrial plant Arabidopsis thaliana revealed that it can also use exogenous cHET, but notably, picoeukaryotic marine phytoplankton and E. coli were adapted to grow on low (picomolar) concentrations of exogenous cHET. Our results call for the modification of the conventional B1 biosynthesis model to incorporate cHET as a key precursor for B1 biosynthesis in two domains of life, and for consideration of cHET as a microbial micronutrient currency modulating marine primary productivity and community interactions in human gut-hosted microbiomes

Nitrogen fixation in the widely distributed marine γ-proteobacterial diazotroph Candidatus Thalassolituus haligoni

International audienceThe high diversity and global distribution of heterotrophic bacterial diazotrophs (HBDs) in the ocean has recently become apparent. However, understanding the role these largely uncultured microorganisms play in marine N 2 fixation poses a challenge due to their undefined growth requirements and the complex regulation of the nitrogenase enzyme. We isolated and characterized Candidatus Thalassolituus haligoni, a member of a widely distributed clade of HBD belonging to the Oceanospirillales. Analysis of its nifH gene via amplicon sequencing revealed the extensive distribution of Cand. T. haligoni across the Pacific, Atlantic, and Arctic Oceans. Pangenome analysis indicates that the isolate shares &gt;99% identity with an uncultured metagenome-assembled genome called Arc-Gamma-03, recently recovered from the Arctic Ocean. Through combined genomic, proteomic, and physiological approaches, we confirmed that the isolate fixes N 2 gas. However, the mechanisms governing nitrogenase regulation in Cand. T. haligoni remain unclear. We propose Cand. T. haligoni as a globally distributed, cultured HBD model species within this understudied clade of Oceanospirillales