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Biology subjects

Markusic, B.

Publications and source records attributed to Markusic, B..

2 recordsLinked to original sources

Contact-phagy remodels ER-mitochondria contact sites

Membrane contact sites between the endoplasmic reticulum (ER) and other organelles coordinate cellular organization, metabolism and stress responses, yet how these interfaces are selectively remodeled and removed remains poorly understood. Here we identify a selective autophagy pathway that targets defined ER-mitochondria contact-site (ERMCS) subdomains for turnover, a process we term Contact-phagy. The ER-resident chaperone SIGMAR1 forms a functional complex with the ER-phagy receptor CCPG1, and SIGMAR1-CCPG1 nanoclusters define ERMCS-associated sites of autophagy initiation. Contact-phagy is selectively engaged by metabolic and mitochondrial stress and promotes turnover of defined contact-site components without broadly affecting general ER-phagy, mitophagy or bulk autophagy. At these sites, SIGMAR1-CCPG1 function is coupled to mitochondrial fission: MFF promotes DRP1 recruitment to ERMCS, whereas loss of CCPG1 or SIGMAR1 causes abnormal persistence and clustering of DRP1 and MFF, accompanied by altered mitochondrial remodeling. Loss of CCPG1 further disrupts ER-mitochondria organization and mitochondrial function in pancreatic tissue, while pancreatic cancer cells depend on CCPG1 and SIGMAR1 for organelle integrity and bioenergetic capacity. Together, our findings establish Contact-phagy as a selective pathway for the turnover of defined organelle contact site subdomains and suggest a broader principle by which interorganelle interfaces are maintained.

cell biology↗

Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up to one-third of all cancers. When BRAF interacts with RAS via the RAS binding domain (RBD) and membrane recruitment, BRAF undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. Despite the central role of BRAF in cellular signaling, the exact order and magnitude of its activation steps has yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation. We employed pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the roles of the regulatory regions in BRAF activation and autoinhibition. Our results demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activity of BRAF. Moreover, we quantified the autoinhibitory binding affinities between the N-terminal domains and the kinase domain (KD) of BRAF and revealed the individual roles of the BRAF regulatory domains. Additionally, our findings provide evidence that the BSR negatively regulates BRAF activation in a RAS isoform-specific manner. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the regulatory domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insights into the activation mechanism of BRAF kinase and may help to guide the development of new therapeutic strategies for cancer treatment.

biochemistry↗