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PPTC7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass and metabolic capacity with elevated hepatic triglyceride accumulation. Pptc7 knockout animals exhibit increased expression of the mitophagy receptors BNIP3 and NIX, and Pptc7 -/- mouse embryonic fibroblasts (MEFs) display a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs, including multiple sites on BNIP3 and NIX, and our molecular studies demonstrate that PPTC7 can directly interact with and dephosphorylate these proteins. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that PPTC7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for PPTC7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function. The mitochondrial phosphatase PPTC7 has previously been linked to the maintenance of mitochondrial content, but the mechanisms underlying this phenotype remain unclear. Here, the authors demonstrate that loss of Pptc7 results in metabolic defects and further suggest that PPTC7 is a regulator of receptor-mediated mitophagy.

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7 −/ − mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7 −/− tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition. The mitochondria houses several phosphatases, but their function is not well characterized. Here, the authors show that mitochondrial phosphatase Pptc7 is important during development for proper mitochondrial function and has a role regulating protein import with the translocase subunit Timm50.

Dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and dietary protein restriction extends the lifespan and healthspan of mice. In this study, we examined the effect of protein restriction (PR) on metabolic health and the development and progression of Alzheimer’s disease (AD) in the 3xTg mouse model of AD. Here, we show that PR promotes leanness and glycemic control in 3xTg mice, specifically rescuing the glucose intolerance of 3xTg females. PR induces sex-specific alterations in circulating and brain metabolites, downregulating sphingolipid subclasses in 3xTg females. PR also reduces AD pathology and mTORC1 activity, increases autophagy, and improves the cognition of 3xTg mice. Finally, PR improves the survival of 3xTg mice. Our results suggest that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD. There is growing need for ways to slow or prevent Alzheimer’s disease (AD). Here, the authors demonstrate that a low protein diet can protect against metabolic dysfunction, slow AD progression, and preserve cognitive function in a mouse model of AD.

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success – including funding, number of publications or journals published in – were unable to completely differentiate applicants with and without job offers. Respondents also reported that the hiring process was unnecessarily stressful, time-consuming, and lacking in feedback, irrespective of outcome. Our findings suggest that there is considerable scope to improve the transparency of the hiring process.

PPTC7 is a mitochondrial-localized phosphatase that suppresses BNIP3- and NIX-mediated mitophagy, but the mechanisms underlying this regulation remain ill-defined. Here, we demonstrate that loss of PPTC7 upregulates BNIP3 and NIX post-transcriptionally and independent of HIF-1α stabilization. Loss of PPTC7 prolongs the half-life of BNIP3 and NIX while blunting their accumulation in response to proteasomal inhibition, suggesting that PPTC7 promotes the ubiquitin-mediated turnover of BNIP3 and NIX. Consistently, overexpression of PPTC7 limits the accumulation of BNIP3 and NIX protein levels, which requires an intact catalytic motif but is surprisingly independent of its targeting to mitochondria. Consistently, we find that PPTC7 is dual-localized to the outer mitochondrial membrane and the matrix. Importantly, anchoring PPTC7 to the outer mitochondrial membrane is sufficient to blunt BNIP3 and NIX accumulation, and proximity labeling and fluorescence co-localization experiments demonstrate that PPTC7 dynamically associates with BNIP3 and NIX within the native cellular environment. Collectively, these data reveal that a fraction of PPTC7 localizes to the outer mitochondrial membrane to promote the proteasomal turnover of BNIP3 and NIX, limiting basal mitophagy.

The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

Liquid chromatography–mass spectrometry (LC–MS) delivers sensitive peptide analysis for proteomics but requires extensive analysis time, reducing throughput. Here, we demonstrate that gas-phase peptide separation instead of LC enables fast proteome analysis. Using direct infusion–shotgun proteome analysis (DI-SPA) by data-independent acquisition mass spectrometry (DIA-MS), we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins per second). We show the utility of this technology in performing a complex multifactorial proteomic study of interactions between nutrients, genotype and mitochondrial toxins in a collection of cultured human cells. More than 45,000 quantitative protein measurements from 132 samples were achieved in only ~4.4 h of MS data collection. Enabling fast, unbiased proteome quantification without LC, DI-SPA offers an approach to boost throughput, critical to drug and biomarker discovery studies that require analysis of thousands of proteomes. Direct infusion–shotgun proteome analysis (DI-SPA) using data-independent acquisition mass spectrometry (DIA-MS) achieves fast and reproducible results by omitting the liquid-chromatography fractionation step and directly performing gas-phase peptide fractionation by ion mobility.

Protein Tyrosine Phosphatase localized to the Mitochondrion 1 (PTPMT1) is a dual specificity phosphatase exclusively localized to the mitochondria, and has recently been shown to be a critical component in the cardiolipin biosynthetic pathway. The downregulation of PTPMT1 in pancreatic beta cells has been shown to increase cellular ATP levels and insulin production, however, the generalized role of PTPMT1 in cancer cells has not been characterized. Here we report that downregulation of PTPMT1 activity is sufficient to induce apoptosis of cancer cells. Additionally, the silencing of PTPMT1 decreases cardiolipin levels in cancer cells, while selectively increasing ATP levels in glycolytic media. Additionally, sublethal downregulation of PTPMT1 synergizes with low doses of paclitaxel to promote cancer cell death. Our data suggest that inhibition of PTPMT1 causes a metabolic crisis in cancer cells that induces cell death, and may be a mechanism by which cancer cells can be sensitized to currently available therapies.

We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.

Mentorship from experienced peers critically improves individual career development and satisfaction in academia, but we have little information on how researchers are supported. We identify and recommend strategies for faculty members, departments, institutions and funders to ensure sustained excellence in academic mentorship.