Molly Plovanich

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter

Mitochondria from diverse organisms are capable of transporting large amounts of Ca2+ via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter’s biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call ‘mitochondrial calcium uniporter’ (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca2+ uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca2+ uniporter.

MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each others protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science, this issue p. 188 A possible sensor for detecting and signaling amino acid abundance is identified. [Also see Perspective by Abraham] The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Reconstitution of the mitochondrial calcium uniporter in yeast

Significance The mitochondrial uniporter is a highly selective calcium channel found in many diverse eukaryotes, but absent in the yeast Saccharomyces cerevisiae. Although the uniporter’s existence was recognized more than 50 y ago, its molecular components have been identified only recently. Here we use yeast as a facile reconstitution system to identify the minimal components sufficient for in vivo uniporter activity. We describe the simplified calcium uniporter of slime mold, consisting of one transmembrane component, DdMCU, which alone is sufficient for robust calcium uptake in yeast mitochondria. Intriguingly, the human uniporter requires two proteins, MCU and the animal-specific protein EMRE, that together are sufficient for uniporter activity. Our work provides a powerful reconstitution system for studying the evolution and function of this channel. The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits –mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)– and two soluble regulatory components–MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

Low Usefulness of Potassium Monitoring Among Healthy Young Women Taking Spironolactone for Acne

IMPORTANCE Spironolactone has been shown to be an effective treatment option for hormonally mediated acne but can cause hyperkalemia. The prevalence of hyperkalemia among healthy young women taking spironolactone for acne is unclear. OBJECTIVE To measure the rate of hyperkalemia in healthy young women taking spironolactone for acne or for an endocrine disorder with associated acne. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of healthy young women taking spironolactone for acne. Data from December 1, 2000, through March 31, 2014, were obtained from a clinical data repository. Outpatient data were collected from 2 tertiary care centers in the United States. We analyzed rates of hyperkalemia in 974 healthy young women taking spironolactone for acne. We also analyzed 1165 healthy young women taking and not taking spironolactone to obtain a profile for the baseline rate of hyperkalemia in this population. Exclusion criteria were cardiovascular disease, renal failure, and the use of medications that affect the renin-angiotensin-aldosterone system. MAIN OUTCOMES AND MEASURES The rate of hyperkalemia in healthy young women taking spironolactone for acne was calculated. Secondary measures included spironolactone prescriber profiles and potassium monitoring practices. RESULTS There were 13 abnormal serum potassium measurements in 1802 measurements obtained among young women receiving spironolactone therapy, yielding a hyperkalemia rate of 0.72%, equivalent to the 0.76% baseline rate of hyperkalemia in this population. Repeat testing in 6 of 13 patients demonstrated normal values, suggesting that these measurements may have been erroneous. In the remaining 7 patients, no action was taken. CONCLUSIONS AND RELEVANCE The rate of hyperkalemia in healthy young women taking spironolactone for acne is equivalent to the baseline rate of hyperkalemia in this population. Routine potassium monitoring is unnecessary for healthy women taking spironolactone for acne.

Novel oral anticoagulants: what dermatologists need to know.

The development of novel oral anticoagulants provides clinicians and patients a welcome alternative to the challenges of warfarin therapy. Dermatologists must be aware of the potential impact of novel oral anticoagulants on their surgical and medical practice. This review provides a concise summary of the novel oral anticoagulants for dermatologists with particular emphasis on: (1) the pharmacokinetic properties of these drugs and how they differ from warfarin, (2) suggested management during cutaneous surgery, (3) adverse drug interactions with commonly prescribed medications in dermatology, and (4) potential use within dermatology for treatment of disorders of cutaneous thrombosis.

The amino acid transporter SLC38A9 is a key component of a lysosomal membrane complex that signals arginine sufficiency to mTORC1

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science, this issue p. 188 A possible sensor for detecting and signaling amino acid abundance is identified. [Also see Perspective by Abraham] The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Giant Pyogenic Granuloma in a Patient with Chronic Lymphocytic Leukemia

Pyogenic granuloma, also known as lobular capillary hemangioma, is a common benign vascular proliferative lesion that can present at any age on the skin or mucous membranes. Most lesions do not exceed 2 cm, but there are a handful of giant cutaneous pyogenic granulomas that have been reported, often in individuals with underlying immune dysfunction. Here, we report the first giant pyogenic granuloma in a patient with a hematological malignancy, chronic lymphocytic leukemia.

Grading and Treatment of Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD) remains a major complication and is the leading cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The most commonly involved organ is the skin, providing an opportunity for clinicians to recognize and intervene in this potentially fatal process. Appropriate grading of severity is critical to determine treatment. Therapy for advanced aGVHD is challenging. This chapter reviews the evidence for commonly studied and novel approaches that hold promise.

Erythematous Rash Following Hematopoietic Stem Cell Transplantation

A 66-year-old man with newly diagnosed multiple myeloma underwent autologous hematopoietic stem cell transplantation (HSCT). On posttransplant day 10, he developed fever (38.4°C). He was started on empirical treatment with cefepime; however, his fevers persisted despite antibiotic therapy. On posttransplant day 14 he developed new-onset pruritic, confluent, erythematous, blanching morbilliform macules and papules on his trunk and extremities (Figure, left). Review of systems was notable for diarrhea and a 6-pound weight gain since transplantation and negative for headache, cough, shortness of breath, or abdominal pain. Onexamination,therewasnolymphadenopathyorhepatosplenomegaly.Thepatient’sheart rate was 100/min, blood pressure was 106/68 mm Hg, and he occasionally experienced mild hypoxiato94%onambientair.Resultsofabasicmetabolicpanelwerenormal,withabloodurea nitrogenlevelof10mg/dL(3.57mmol/L)andcreatininelevelof0.7mg/dL(61.88μmol/L).White blood cell (WBC) count was 7300/μL, and absolute neutrophil count (ANC) was 5480/μL. Neutrophil recovery occurred beginning on posttransplant day 10, with WBC count of 700/μL and ANC of 532/μL on posttransplant day 10, correlating with the onset of the patient’s fevers. Platelet count was 48 × 103/μL, and hemoglobin was 8.2 g/dL. Results of liver function tests were normal. Blood, urine, sputum, and respiratory viral cultures were negative. Tests for cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, adenovirus, parvovirus, cryptococcal antigen, aspergillus galactomannan antigen, β-glucan, and Clostridium difficile were negative. Chest computed tomography demonstrated multifocal ground-glass and consolidative pulmonary opacities (Figure, right). Computed tomography of the abdomen was unremarkable. A skin punch biopsy demonstrated subtle vacuolar change; rare epidermal dyskeratosis, including within a hair follicle; and superficial perivascular mononuclear cell infiltrate with occasional eosinophils.