A mutation in the low-complexity domain of splicing factor hnRNPA1 linked to amyotrophic lateral sclerosis disrupts distinct neuronal RNA splicing networks.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

An interim exploratory proteomics biomarker analysis of a phase 2 clinical trial to assess the impact of CT1812 in Alzheimer's disease.

CT1812 is a novel, brain penetrant small molecule modulator of the sigma-2 receptor (S2R) that is currently in clinical development for the treatment of Alzheimer's disease (AD). Preclinical and early clinical data show that, through S2R, CT1812 selectively prevents and displaces binding of amyloid beta (Aß) oligomers from neuronal synapses and improves cognitive function in animal models of AD. SHINE is an ongoing phase 2 randomized, double-blind, placebo-controlled clinical trial (COG0201) in participants with mild to moderate AD, designed to assess the safety and efficacy of 6 months of CT1812 treatment. To elucidate the mechanism of action in AD patients and pharmacodynamic biomarkers of CT1812, the present study reports exploratory cerebrospinal fluid (CSF) biomarker data from 18 participants in an interim analysis of the first set of patients in SHINE (part A). Untargeted mass spectrometry-based discovery proteomics detects >2000 proteins in patient CSF and has documented utility in accelerating the identification of novel AD biomarkers reflective of diverse pathophysiologies beyond amyloid and tau, and enabling identification of pharmacodynamic biomarkers in longitudinal interventional trials. We leveraged this technique to analyze CSF samples taken at baseline and after 6 months of CT1812 treatment. Proteome-wide protein levels were detected using tandem mass tag-mass spectrometry (TMT-MS), change from baseline was calculated for each participant, and differential abundance analysis by treatment group was performed. This analysis revealed a set of proteins significantly impacted by CT1812, including pathway engagement biomarkers (i.e., biomarkers tied to S2R biology) and disease modification biomarkers (i.e., biomarkers with altered levels in AD vs. healthy control CSF but normalized by CT1812, and biomarkers correlated with favorable trends in ADAS-Cog11 scores). Brain network mapping, Gene Ontology, and pathway analyses revealed an impact of CT1812 on synapses, lipoprotein and amyloid beta biology, and neuroinflammation. Collectively, the findings highlight the utility of this method in pharmacodynamic biomarker identification and providing mechanistic insights for CT1812, which may facilitate the clinical development of CT1812 and enable appropriate pre-specification of biomarkers in upcoming clinical trials of CT1812.

TMT-MS/MS proteomic analysis of the carbohydrate-active enzymes in the fruiting body of Pleurotus tuoliensis during storage.

BACKGROUND: The fruiting body of Pleurotus tuoliensis deteriorates rapidly after harvest, causing a decline in its commercial value and a great reduction in its shelf life. According to the present research, carbohydrate-active enzymes (CAZymes) may cause the softening, liquefaction and autolysis of mature mushrooms after harvest. To further understand the in vivo molecular mechanism of CAZymes affecting the postharvest quality of P. tuoliensis fruiting bodies, a tandem mass tags labelling combined liquid chromatography-tandem mass spectrometry (TMT-MS/MS) proteomic analysis was performed on P. tuoliensis fruiting bodies during storage at 25 °C. RESULTS: A total of 4737 proteins were identified, which had at least one unique peptide and had a confidence level above 95%. Consequently, 1307 differentially expressed proteins (DEPs) were recruited using the criteria of abundance fold change (FC) >1.5 or < 0.67 and P < 0.05. The identified proteins were annotated by dbCAN2, a meta server for automated CAZymes annotation. Subsequently, 222 CAZymes were obtained. Several CAZymes participating in the cell wall degradation process, including ß-glucosidase, glucan 1,3-ß-glucosidase, endo-1,3(4)-ß-glucanase and chitinases, were significantly upregulated during storage. The protein expression level of CAZymes, such as xylanase, amylase and glucoamylase, were upregulated significantly, which may participate in the P. tuoliensis polysaccharide degradation. CONCLUSIONS: The identified CAZymes degraded the polysaccharides and lignin, destroying the cell wall structure, preventing cell wall remodeling, causing a loss of nutrients and the browning phenomenon, accelerating the deterioration of P. tuoliensis fruiting body. © 2020 Society of Chemical Industry.

Pseudomonas aeruginosa Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung.

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2 < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease.

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student ('Case'), who died of IMD, and a close contact ('Carrier') who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.