Project description:FAM134B is a reticulon-homology domain (RHD)-containing protein that participates in membrane-shaping of the endoplasmic reticulum (ER)8 13. It also functions as a mammalian ER-phagy receptor, mediating the fragmentation and selective degradation of ER sheets in multiple cell types8. However, little is known about the molecular and biophysical mechanisms that control and/or switch between these two FAM134B functions.
Project description:Stop codon recoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene.
In order to systematically assess the frequency and origin of recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur as high as 80 percent of the time, depending on the genetic context, suggesting that evolution frequently samples stop codon recoding events. Targeted mass spectrometry and RNA-seq analyses showed that not only translational but also transcriptional errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide mass -spectrometry revealed that temperature regulates the expression of cryptic peptides generated by stop codon recoding in E.coli.
Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.
Project description:A ELMSeq reporter cassette was created to monitor Dam levels by methylation, and introduced in the genome. The regions of 6 nt upstream and 6 nt downstream the stop codon were randomized to study their effect on gene expression. The ELMSeq reporter cassette was composed of: promoter - dam - random N6 - stop codon TAA - random N6 - spacer - 4xGATC. The amplicon was spanning the C-terminal region. The cassette was introduced in Mycoplasma pneumoniae.
Project description:Rheumatic heart disease (RHD) remains a major source of morbidity and mortality in developing countries. A deeper insight into the pathogenetic mechanisms underlying RHD could provide opportunities for drug repurposing, guide recommendations for secondary penicillin prophylaxis, and/or inform development of near-patient diagnostics. We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to screen protein expression in 215 African patients with severe RHD, and 230 controls. A machine learning (ML) approach was applied to feature selection among the 366 proteins quantifiable in at least 40% of samples, using the Boruta wrapper algorithm. The case-control differences and contribution to AUC of the ROC for each of the 56 proteins identified by the Boruta algorithm were calculated by Logistic Regression adjusted for age, sex and BMI. Adiponectin, complement component C7 and fibulin-1, a component of heart valve matrix, were each higher in cases when compared with controls. Ficolin-3, a protein with calcium-independent lectin activity that activates the complement pathway, was lower in cases than controls. The top six biomarkers from the Boruta analyses conferred an AUC of 0.90 indicating excellent discriminatory capacity between RHD cases and controls.
