Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic F e-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes. Keywords: LYR; ACP; iron-sulfur cluster; PLP; frataxi

Mitochondrial quality control by the ubiquitin-proteasome system

Abstract Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondriaassociated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosincontaining protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells

\u3ci\u3eSDH5\u3c/i\u3e, a Gene Required for Flavination of Succinate Dehydrogenase, Is Mutated in Paraganglioma

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene

\u3ci\u3eSDH5\u3c/i\u3e, a Gene Required for Flavination of Succinate Dehydrogenase, Is Mutated in Paraganglioma

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene

The Role of PAS Kinase in PASsing the Glucose Signal

PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration

Pyruvate metabolism controls chromatin remodeling during CD4+ T cell activation

Upon antigen-specific T cell receptor (TCR) engagement, human CD4 + T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient T cells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, T cell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4 + T cell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven T cell activation

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment