-
Molecular Cell Aug 2023Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate...
Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.
Topics: Humans; Acclimatization; Mitochondria; Saccharomyces cerevisiae; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Mitochondrial Proteins
PubMed: 37595558
DOI: 10.1016/j.molcel.2023.07.021 -
Nature Aug 2023Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation. The Rpd3 small (Rpd3S) complex recognizes histone H3...
Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.
Topics: Acetylation; Cryoelectron Microscopy; DNA, Fungal; Epigenesis, Genetic; Histones; Lysine; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Methylation; Multiprotein Complexes
PubMed: 37468628
DOI: 10.1038/s41586-023-06349-1 -
Proceedings (Baylor University. Medical... 2023Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is an autoimmune condition characterized by necrotizing inflammation of small vessels throughout the...
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is an autoimmune condition characterized by necrotizing inflammation of small vessels throughout the body. Pharmaceutical agents have been noted as an emerging etiology. This case presents a 41-year-old woman with a longstanding history of Graves' disease who previously failed other interventions and was started on propylthiouracil (PTU) nearly 2 years prior to symptom onset. The patient presented with severely pruritic purpuric lesions on her lower extremities that transformed into large bullae and became extremely painful. A thorough workup revealed only slightly elevated perinuclear ANCA and a mild protein S deficiency. Tissue biopsy was consistent with thrombotic vasculitis. A presumptive clinical diagnosis of PTU-induced vasculitis was made. Because the condition is relatively uncommon, the best course of treatment has not clearly been defined. Though PTU was immediately discontinued, the patient also required corticosteroids and referral for tissue debridement. While some cases have had symptom resolution after cessation of PTU, this case adds to a growing body of evidence for the timely use of corticosteroids in controlling PTU-induced vasculitis.
PubMed: 37614859
DOI: 10.1080/08998280.2023.2230830 -
BioRxiv : the Preprint Server For... Jul 2023Amino acid mutations that lower a protein's thermodynamic stability are implicated in numerous diseases, and engineered proteins with enhanced stability are important in...
Amino acid mutations that lower a protein's thermodynamic stability are implicated in numerous diseases, and engineered proteins with enhanced stability are important in research and medicine. Computational methods for predicting how mutations perturb protein stability are therefore of great interest. Despite recent advancements in protein design using deep learning, prediction of stability changes has remained challenging, in part due to a lack of large, high-quality training datasets for model development. Here we introduce ThermoMPNN, a deep neural network trained to predict stability changes for protein point mutations given an initial structure. In doing so, we demonstrate the utility of a newly released mega-scale stability dataset for training a robust stability model. We also employ transfer learning to leverage a second, larger dataset by using learned features extracted from a deep neural network trained to predict a protein's amino acid sequence given its three-dimensional structure. We show that our method achieves competitive performance on established benchmark datasets using a lightweight model architecture that allows for rapid, scalable predictions. Finally, we make ThermoMPNN readily available as a tool for stability prediction and design.
PubMed: 37547004
DOI: 10.1101/2023.07.27.550881 -
Cancer Research Apr 2024Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to...
UNLABELLED
Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1-dependent methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phosphoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied α-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacologic inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, IHC coupled with imaging MS in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC.
SIGNIFICANCE
PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.
Topics: Humans; Phosphoglycerate Dehydrogenase; Triple Negative Breast Neoplasms; Drug Resistance, Neoplasm; Serine; Palmitates; Fatty Acids; Cell Line, Tumor; Protein-Arginine N-Methyltransferases; Repressor Proteins
PubMed: 38383964
DOI: 10.1158/0008-5472.CAN-23-2266 -
Journal of Orthopaedic Translation Sep 2023Excessive osteoclast formation disrupts bone homeostasis, thereby significantly contributing to pathological bone loss associated with a variety of diseases. Protein...
BACKGROUND
Excessive osteoclast formation disrupts bone homeostasis, thereby significantly contributing to pathological bone loss associated with a variety of diseases. Protein S-palmitoylation is a reversible post-translational lipid modification catalyzed by ZDHHC family of palmitoyl acyltransferases, which plays an important role in various physiological and pathological processes. However, the role of palmitoylation in osteoclastogenesis has never been explored. Consequently, it is unclear whether this process can be targeted to treat osteolytic bone diseases that are mainly caused by excessive osteoclast formation.
MATERIALS AND METHODS
In this study, we employed acyl-biotin exchange (ABE) assay to reveal protein S-palmitoylation in differentiating osteoclasts (OCs). We utilized 2-bromopalmitic acid (2-BP), a pharmacological inhibitor of protein S-palmitoylation, to inhibit protein palmitoylation in mouse bone marrow-derived macrophages (BMMs), and tested its effect on receptor activator of nuclear factor κβ ligand (RANKL)-induced osteoclast differentiation and activity by TRAP staining, phalloidin staining, qPCR analyses, and pit formation assays. We also evaluated the protective effect of 2-BP against estrogen deficiency-induced bone loss and bone resorption in ovariectomized (OVX) mice using μCT, H&E staining, TRAP staining, and ELISA assay. Furthermore, we performed western blot analyses to explore the molecular mechanism underlying the inhibitory effect of 2-BP on osteoclastogenesis.
RESULTS
We found that many proteins were palmitoylated in differentiating OCs and that pharmacological inhibition of palmitoylation impeded RANKL-induced osteoclastogenesis, osteoclast-specific gene expression, F-actin ring formation and osteoclastic bone resorption , and to a lesser extent, osteoblast formation from MC3T3-E1 cells. Furthermore, we demonstrated that administration of 2-BP protected mice from ovariectomy-induced osteoporosis and bone resorption . Mechanistically, we showed that 2-BP treatment inhibited osteoclastogenesis partly by downregulating the expression of c-Fos and NFATc1 without overtly affecting RANKL-induced activation of osteoclastogenic AKT, MAPK, and NF-κB pathways.
CONCLUSION
Pharmacological inhibition of palmitoylation potently suppresses RANKL-mediated osteoclast differentiation and protects mice against OVX-induced osteoporosis Mechanistically, palmitoylation regulates osteoclast differentiation partly by promoting the expression of c-Fos and NFATc1. Thus, palmitoylation plays a key role in promoting osteoclast differentiation and activity, and could serve as a potential therapeutic target for the treatment of osteoporosis and other osteoclast-related diseases.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE
The translation potential of this article is that we first revealed palmitoylation as a key mechanism regulating osteoclast differentiation, and therefore provided a potential therapeutic target for treating osteolytic bone diseases.
PubMed: 37521493
DOI: 10.1016/j.jot.2023.06.002 -
Arteriosclerosis, Thrombosis, and... Jul 2023Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of...
BACKGROUND
Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total.
METHODS
Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes.
RESULTS
Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (, , and ) and 1 with PS levels (-). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (), 1 with PC (), and 1 with PS (). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of , , and genes in antithrombin regulation.
CONCLUSIONS
The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.
Topics: Protein C; Protein S; Genome-Wide Association Study; Antithrombins; Transcriptome; Anticoagulants; Antithrombin III; Polymorphism, Single Nucleotide
PubMed: 37128921
DOI: 10.1161/ATVBAHA.122.318213