Project description:Mammalian fatty acid synthase (FASN) is a lipogenic enzyme that catalyzes the formation of the long chain saturated fatty acid palmitate from acetyl and malonyl CoA in the presence of NADPH. Mammalian cells acquire fatty acids through dietary sources or through FASN. Although most mammalian cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (5-Hexynoic acid, or "Alk-4") that functions as a reporter of FASN-dependent protein acylation. Alk-4 metabolic labeling enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Alk-4 also labeled the palmitoylated host protein IFITM3 (Interferon inducible transmembrane protein-3), a restriction factor for Influenza, and the myristoylated HIV-1 MA (Matrix) protein. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.
Project description:Mitochondrial function is modulated by its functional interaction with the endoplasmic reticulum. Recent research indicates that these contacts are disrupted in familial models of amyotrophic lateral sclerosis. We report here this impairment in the crosstalk between mitochondria and the endoplasmic reticulum impedes the use of glucose-derived pyruvate as mitochondrial fuel, causing a shift to fatty acids to sustain energy production. Over time, this deficiency alters mitochondrial electron flow and the active/dormant status of complex I in spinal cord tissues, but not in the brain. These findings suggest MAM plays a crucial role in regulating cellular glucose metabolism and that its dysfunction may underlie the bioenergetic deficits observed in ALS.
Project description:Pancreatic cancer (PC) remains one of the most aggressive and life-threatening malignancies known for its notorious resistance to chemotherapy. This is increasingly ascribed to the subpopulation of undifferentiated cells, known as pancreatic cancer stem cells (PCSCs), which are evolutionary fitter than other tumor cells to evade the cytotoxic effects of chemotherapy. Those cells are crucial for tumor relapse as they possess ‘stem cell-like’ features of self-renewal and differentiation. However, what molecular mechanisms maintain the unique characteristics of PCSCs are poorly understood. Here, we identified an RNA polymerase II-associated PHF5A-PHF14-HMG20A-RAI1-KMT2A transcriptional subcomplex, which regulates the stemness characteristics and tumorigenicity of PCSCs through epigenetic control of gene expression. Targeting the protein subcomplex with a KMT2A-WDR5 inhibitor attenuated the self-renewal and in vivo tumorigenicity of PCSCs, thus offering a novel anti-PCSCs targeting strategy for enhancing the efficiency of chemotherapy which is likely to translate into durable clinical responses in PC patients.
Project description:We previously reported a pathogenic de novo W342 mutation in the transcriptional corepressor CtBP1 in four independent patients with neurodevelopmental disabilities. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CtBP1 mutation. Within this cohort we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia and tooth enamel defects present in all patients. The W342 mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cells lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.
Project description:To uncover putative RPM-1 protein-protein interactions, we performed a comprehensive series of in vivo affinity-purification proteomics experiments using C. elegans. We utilized integrated transgenes that express RPM-1 fused with a Protein G::Streptavidin binding peptide (GS) affinity tag expressed using the native rpm-1 promoter on an rpm-1 protein null background. Negative control animals expressed an integrated transgene where the GS tag was fused to GFP (GS::GFP) and expressed by the rpm-1 promoter. We also designed a biochemical “trap” using an RPM-1 ligase-dead (RPM-1 LD) site-directed mutant to identify ubiquitination substrates. RPM-1 LD would enrich substrates by trapping them in stalled ubiquitination complexes, as well as preventing proteasome-mediated degradation and elevating substrate levels. RPM-1-binding proteins that are not ubiquitination substrates would bind to both RPM-1 and RPM-1 LD.
Project description:Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells for diverse roles in antiviral defence. Here we determine structures of a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease and four molecules of an SMC-family ATPase assemble into a 364 kDa complex that provides phage defence. We show that phage-encoded nucleases trigger degradation of the retron-associated DNA, leading to disassembly of the retron and activation of the HNH nuclease. The HNH nuclease cleaves tRNASer, stalling protein synthesis and arresting viral replication. Taken together, these data reveal diverse and paradoxical roles for RTs in the perpetuation and elimination of genetic parasites.
Project description:Mono-ubiquitinated PCNA (mono-Ub-PCNA) is generated when replication forks stall and facilitates the DNA lesion bypass process. After resolving a replication stall, Ub-PCNA needs to be de-ubiquitinated to resume high-fidelity DNA synthesis. ATAD5 cooperates with UAF1-USP1 to de-ubiquitinate mono-Ub-PCNA. However, it remains unclear how Ub-PCNA de-ubiquitination is regulated in a timely manner. We found that BAZ1B, a regulatory subunit of the chromatin-remodeling complex, fine-tunes de-ubiquitination of Ub-PCNA. The BAZ1B binding region of ATAD5 surrounds the ATAD5 UAF1-binding domain. Abrogation of the ATAD5-BAZ1B interaction leads to premature de-ubiquitination of Ub-PCNA after hydrogen peroxide treatment. BAZ1B-binding defective ATAD5 cells are more sensitive to oxidative stress compared to wild-type cells. These results suggest that BAZ1B inhibits premature Ub-PCNA de-ubiquitination to maintain genome integrity.
Project description:The DNA exonuclease TREX1 degrades endogenous cytosolic DNA. Cytosolic DNA triggers the cGAS/STING pathway which increases type I interferon. To investigate the physiological significance of TREX1 loss on in vivo tumor growth, we implanted control and TREX1-deficient CT26 tumor cells into immunocompetent BALB/c hosts.Tumor cells were collected 7 days after tumors reached around 200mm3.
Project description:Aminoacyl-tRNA synthetases (aaRSs) catalyze the ligation of each amino acid to the 3’ hydroxyl group of the cognate tRNA and thereby establish the genetic code for protein synthesis. AaRSs form a complicated network through assembly into a multi-synthetase complex (MSC), which is comprised of nine aaRSs (ArgRS, AspRS, GlnRS, GluProRS, IleRS, LeuRS, LysRS, and MetRS) and three auxiliary proteins (aaRS-interacting multifunctional proteins, p43, p38, and p18 or AIMP1, 2, and 3) in vertebrates. IleRS is one of the least characterized aaRSs in the MSC. It is a class 1a aaRS and has an UNE-I domain at its C-terminal end that is formed by tandem ~90 residue repeats. Systematic depletion and yeast two-hybrid (Y2H) analyses suggest that IleRS binds to the WHEP domain of GluProRS through its UNE-I domain. However, crosslinking and mass spectrometry (XL-MS) analyses revealed that IleRS interacts with ArgRS, LeuRS, and MetRS in the MSC. While such difference may be due to the different dynamic states of the MSC under various cellular environments, it remains to be resolved how IleRS associates with other components of the MSC. Therefore, we performed mass spectrometry analysis to study IleRS interactome using cells expressing wild-type IleRS, C-terminal-deleted IleRS, and the IleRS UNE-I domain alone.
Project description:This project is a report of a chromosome-based human proteome project focused on chromosome 9 (Chr 9). To reveal missing proteins and undiscovered features in proteomes, LC-MS/MS analysis based identification and characterization were conducted on 5 pairs of lung adenocarcinoma tumors and adjacent non-tumor tissues.