Biology subjects

Hajnoczky, G.

Publications and source records attributed to Hajnoczky, G..

4 recordsLinked to original sources

bioRxiv2024

Transcriptional regulation in the absence of inositol trisphosphate receptor calcium signaling

The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that regulate a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB, AP-1 and NF{kappa}b) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition the role of protein kinase C (PKC) was investigated with inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression (DEG) of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. In summary three main adaptations in TKO cells are identified in this study: 1) increased basal activity of NFAT, CREB, AP-1 and NF{kappa}b; 2) an increased reliance on Ca2+-insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g. PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.

cell biology↗

bioRxiv2024

Systematic mapping of MCU-mediated mitochondrial calcium signaling networks

The Mitochondrial Ca2+ Uniporter Channel (MCUC) allows calcium entry into the mitochondrial matrix to regulate energy metabolism but also cell death. Although, several MCUC components have been identified, the molecular basis of mitochondrial Ca2+ signaling networks and their remodeling upon changes in uniporter activity have not been systematically assessed. Using an unbiased and quantitative proteomic approach, we map the MCUC interactome in HEK293 cells under physiological conditions and upon chronic loss or gain of mitochondrial Ca2+ uptake. Besides all previously known subunits of the uniporter, we identify 89 high-confidence interactors linking MCUC to several mitochondrial complexes and pathways, half of which are currently linked to metabolic, neurological, and immunological diseases. As a proof-of-concept, we validate EFHD1 as a binding partner of MCU, EMRE and MCUB with a MICU1-dependent inhibitory effect on Ca2+ uptake. To investigate compensatory mechanisms and functional consequences of mitochondrial Ca2+ dyshomeostasis, we systematically survey the MCU interactome upon silencing of EMRE, MCUB, MICU1 or MICU2. We observe profound changes in the MCU interconnectivity, whereby downregulation of EMRE reduces the number of MCU interactors of over 10-fold, while silencing of MCUB leads to a wider functional network linking MCU to mitochondrial stress response pathways and cell death. Altogether our study provides a comprehensive map of MCUC protein-protein interactions and a rich, high-confidence resource that can be explored to gain insights into the players and mechanisms involved in calcium signal transduction cascades and their relevance in human diseases.

cell biology↗

bioRxiv2024

Mitochondrial membrane potential regulates nuclear DNA methylation and gene expression through phospholipid remodeling

Maintenance of the mitochondrial inner membrane potential ({Delta}{Psi}M) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While {Delta}{Psi}M loss and its consequences are well studied, little is known about the effects of increased {Delta}{Psi}M. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic {Delta}{Psi}M increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the {Delta}{Psi}M to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the {Delta}{Psi}M, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the {Delta}{Psi}M as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction. HighlightsO_LIMitochondria hyperpolarization leads to nuclear DNA hypermethylation C_LIO_LIDNA methylation regulates expression of mitochondrial and lipid metabolism genes C_LIO_LIPhospholipid remodeling mediates the epigenetic effects of mitochondrial hyperpolarization C_LI

genomics↗

bioRxiv2021

REDUCED ER-MITOCHONDRIA CONNECTIVITY PROMOTES NEUROBLASTOMA MULTIDRUG RESISTANCE

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptotic programs. We isolated mitochondria from neuroblastoma cell lines obtained from children at diagnosis and after relapse following failed therapy, and profiled responses to tBid and Bim, death effectors activated by therapeutic stress. Mitochondria from post-relapse models had markedly attenuated cytochrome c release (surrogate for apoptotic commitment) in comparison with patient-matched diagnostic models. Mitochondrial DNA content, size, and shape did not differ consistently. However, we used electron microscopy to identify reduced endoplasmic reticulum-mitochondria contacts (ERMCs) as correlated with therapy resistance. ERMCs form microdomains for the transfer of Ca2+ to mitochondria. We confirmed reduced Ca2+ transfer in resistant cells, with restoration by re-opposing ERMCs via genetically-encoded linkers. However, reduced Ca2+ transfer was not present in all ERMC-reduced cancers with therapy resistance, supporting Ca2+-independent mechanisms. Genetically or biochemically reducing ERMCs in therapy sensitive tumors phenocopied resistance, validating these inter-organelle contacts as physiologic regulators of apoptosis. Our work confirms the importance of ERMCs in stress signaling and provides a previously unrecognized mechanism for cancer cell resistance that is not exclusive to other contributors.

cancer biology↗