1,721,075 research outputs found
Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans
No full textUpstream ORFs (uORFs) are mRNA elements defined by a start codon in the 5′ UTR that is out-of-frame with the main coding sequence. Although uORFs are present in approximately half of human and mouse transcripts, no study has investigated their global impact on protein expression. Here, we report that uORFs correlate with significantly reduced protein expression of the downstream ORF, based on analysis of 11,649 matched mRNA and protein measurements from 4 published mammalian studies. Using reporter constructs to test 25 selected uORFs, we estimate that uORFs typically reduce protein expression by 30–80%, with a modest impact on mRNA levels. We additionally identify polymorphisms that alter uORF presence in 509 human genes. Finally, we report that 5 uORF-altering mutations, detected within genes previously linked to human diseases, dramatically silence expression of the downstream protein. Together, our results suggest that uORFs influence the protein expression of thousands of mammalian genes and that variation in these elements can influence human phenotype and disease.National Institute of General Medical Scienc
Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging
No full textObtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.close
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Mitochondrial Triggers of the Integrated Stress Response: Disentangling the Bioenergetic Rubik’s Cube
No full textMitochondria are hubs of metabolism and signaling in eukaryotic cells whose dysfunction underlies a class of devastating genetic disorders and is also frequently associated with common conditions, such as neurodegeneration, diabetes, cancer and the ageing process. Mitochondrial dysfunction, particularly breakdown of the electron transport chain (ETC) and oxidative phosphorylation, yields a perplexingly variable spectrum of consequences at the cellular, tissue and whole-organism level. Deciphering the context-dependent pathophysiology of mitochondrial dysfunction is thus a major challenge in both basic and translational biomedical research.
Studies over the past decade have revealed that a prominent molecular signature of mitochondrial dysfunction in vivo is activation of the integrated stress response (ISR), a gene expression program eukaryotic cells engage upon different types of insults. How mitochondrial dysfunction is sensed to trigger the ISR and whether the response serves a protective role or contributes to pathology remain far from understood.
The work in this thesis sought to delineate functional parameters tied to the ETC, such as ATP synthesis or NADH oxidation, that can lead to ISR activation. We used chemical and genetic tools to perturb ETC functions in mouse muscle cells while specifically compensating for some of the resulting metabolic effects. We then monitored the impact of these interventions on ISR-dependent gene expression by RNA sequencing.
Our results revealed that in proliferating cells, the increase in the cytosolic [NADH]/[NAD+] ratio during complex I dysfunction potently triggered the ISR, mostly by sharply depressing aspartate levels and activating the amino acid sensitive eIF2α kinase GCN2. Strikingly, this route to ISR activation became inoperative in terminally-differentiated myotubes where only ATP synthase inhibition elicited a significant response. The path to ISR activation in the latter case was dependent on residual ETC activity and could be abolished by co-inhibition of complex I, mild uncoupling or mild hypoxic preconditioning. Finally, our data suggests dysfunction of mitochondrial genome expression is not directly sensed to trigger the ISR.
These results shed light on the complicated interplay between mitochondrial dysfunction and the ISR. They implicate diverse metabolic and bioenergetic routes to its activation whose relevance in vivo should be carefully evaluated in future work.Systems Biolog
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Translational control through differential ribosome pausing during amino acid limitation in mammalian cells
No full textLimitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that limitation for the amino acid arginine causes a selective loss of isoacceptor tRNA charging, which regulates translation through selective ribosome pausing at two of six arginine codons. Interestingly, limitation for leucine, an essential and abundant amino acid in proteins, resulted in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevented ribosome pausing, while an insufficient response to arginine limitation led to loss of arginine tRNA charging and ribosome pausing. Codon-specific ribosome pausing decreased protein production specifically during arginine limitation without significantly reducing mRNA levels. Together, our results suggest an evolutionarily conserved role for synonymous codon usage in elongation rate control of protein synthesis during limitation for single amino acids.Biology, Molecular and Cellulartranslation elongation, translation initiation, mTORC1, GCN2, amino acid homeostasis, ribosome pausin
A plasma signature of human mitochondrial disease revealed through metabolic profiling of spent media from cultured muscle cells
No full textMutations in either the mitochondrial or nuclear genomes can give rise to respiratory chain disease (RCD), a large class of devastating metabolic disorders. Their clinical management is challenging, in part because we lack facile and accurate biomarkers to aid in diagnosis and in the monitoring of disease progression. Here we introduce a sequential strategy that combines biochemical analysis of spent media from cell culture with analysis of patient plasma to identify disease biomarkers. First, we applied global metabolic profiling to spotlight 32 metabolites whose uptake or secretion kinetics were altered by chemical inhibition of the respiratory chain in cultured muscle . These metabolites span a wide range of pathways and include lactate and alanine, which are used clinically as biomarkers of RCD. We next measured the cell culture-defined metabolites in human plasma to discover that creatine is reproducibly elevated in two independent cohorts of RCD patients, exceeding lactate and alanine in magnitude of elevation and statistical significance. In cell culture extracellular creatine was inversely related to the intracellular phosphocreatine:creatine ratio suggesting that the elevation of plasma creatine in RCD patients signals a low energetic state of tissues using the phosphocreatine shuttle. Our study identifies plasma creatine as a potential biomarker of human mitochondrial dysfunction that could be clinically useful. More generally, we illustrate how spent media from cellular models of disease may provide a window into the biochemical derangements in human plasma, an approach that could, in principle, be extended to a range of complex diseases.Broad Institute. Scientific Planning and Allocation of Resources CommitteeNational Institutes of Health (U.S) (R01DK081457
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Linking Human Genetic Variation to Mitochondrial Metabolism
No full textGenetic variation has a powerful impact on human metabolism and disease. Traditionally, this relationship has either been studied at a high level using top-down descriptive studies of patients with genetically defined inborn errors of metabolism, or else from the bottom up, with molecular biology and biochemical studies of single proteins. Recent advances in genetic sequencing, metabolic profiling technology, and structural biology are rapidly enabling the integration of these approaches towards a more complete description of human metabolism
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Quantitative Approaches to Cellular Information Processing and Metabolic Regulation
No full textOrganisms of all levels of complexity must undertake complex information processing tasks. Diverse cellular and biochemical mechanisms are required to integrate multiple sources of information and to balance performance trade-offs, such as between speed and accuracy or robustness and fragility. This dissertation describes a series of quantitative analyses of cellular information processing, with particular attention given to the regulation of metabolism. Chapters 2 and 3 consider mechanisms for achieving concentration robustness in signal transduction. Chapter 2 develops a large compendium of reaction networks involving bifunctional enzymes, which are often positioned at key metabolic branch points and are experimentally associated with robust control. Using high-throughput algebraic analysis of this compendium, we demonstrate that bifunctional enzymes can implement five different forms of concentration robustness, and that the type of robustness is highly sensitive to biochemical details beyond bifunctionality. Chapter 3 identifies intermediate buffering in a three-component phospho-relay as a novel mechanism for concentration robustness and argues that such a mechanism accounts for robust inactivation of the yeast osmotic stress response. Chapter 4 reports an integrated computational and experimental analysis of production of the oncometabolite 2-hydroxyglutarate by mutant isocitrate dehydrogenase 1 (IDH1), which suggests that the clinically observed retention of a wild-type (WT) IDH1 allele in tumors is not due to a requirement for substrate channeling or substantial inter-subunit flux in WT/mutant IDH1 heterodimers. In Chapter 5 we examine the information processing capabilities of calcium/calmodulin signaling and show that a straightforward equilibrium binding analysis can clarify longstanding questions about the control of smooth muscle contraction. Finally, Chapter 6 reports an experimental approach to investigate the limits of complex information processing in single cells. Resurrecting a classical body of literature on the behavior of unicellular organisms, we demonstrate that the giant ciliate Stentor roeseli engages in multi-step hierarchical sequences of avoidance behaviors. The S. roeseli avoidance response is distinct from other primitive forms of learning such as habituation and conditioning and is suggestive of complex decision-making by the organism. Throughout the dissertation, a common theme is the use of mathematical modeling to link biochemical form to physiological function and to generate experimentally testable predictions that are independent of hard-to-measure parameter values.Systems Biolog
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Regulation of the Mitochondrial Calcium Uniporter by the MICU1/MICU2 Complex: From Biochemistry to Human Disease
No full textThe mitochondrial calcium uniporter is an evolutionarily ancient calcium channel, with purported roles in many areas of cellular and organismal physiology from excitation: energetic coupling to cell death. The molecular components of the uniporter were elusive until 2010, when the inaugural component, MICU1, was identified and the remaining components were identified shortly thereafter. Here, we explore the function of both MICU1 and MICU2, ranging from protein biochemistry to human disease.
We start with protein biochemistry and cellular physiology to define the role of MICU1 and MICU2 in uniporter function. Chapters 2 and 3 unveil the discovery of MICU2 and show that MICU1 and MICU2 are negative regulators of the uniporter in the intermembrane space. In Chapter 4, we show that the calcium binding affinities of MICU1 and the MICU1/MICU2 complex control the calcium concentration needed to disinhibit the uniporter. We provide further support for this model in Chapter 5, showing that MICU1 and MICU2 contribute to the selectivity of the uniporter complex, preventing manganese transport through the channel unless sufficient calcium is present. Together, these data converge on a model in which the apo (calcium-free) MICU1 and MICU2 inhibit the uniporter, keeping the channel closed; when calcium concentrations rise above ~600 nM, calcium binds to MICU1 and MICU2, allowing the channel to open.
In Chapters 6 and 7, we move from biochemistry to organismal physiology. Chapter 6 discusses the cardiovascular phenotype of Micu2 knockout mice. These mice have diastolic dysfunction and, strikingly, when treated with Angiotensin II to induce hypertension, almost one-third of the knockout mice die from abdominal aortic aneurysm rupture. We explore the clinical presentation of two cousins with MICU1 deficiency in Chapter 7. Patients with MICU1 loss-of-function mutations present with a neuromuscular syndrome, including fatigue and lethargy. Calcium physiology experiments in patient fibroblasts reveal a defect in mitochondrial calcium handling suggesting matrix calcium overload, consistent with MICU1 deficiency.
Great progress has been made in the past several years, but many important discoveries are still to come. Structural insights into the functional uniporter channel, the MICU1/MICU2 complex, and ultimately the holocomplex will be critical next steps. Furthermore, we need to better understand the uniporter’s role in physiology and human disease. Synthesizing our knowledge of the uniporter structure, function, and roles in pathophysiology may lead to exciting new therapeutics targeting the uniporter for both rare and common disease.Chemistry and Chemical Biolog
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