TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.

PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling.

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.

Identification of novel genes and pathways regulating SREBP transcriptional activity.

BACKGROUND: Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways. METHODOLOGY: To identify novel regulators of SREBPs, we performed a genome-wide cDNA over-expression screen to identify proteins that might modulate the transcription of a luciferase gene driven from an SREBP-specific promoter. The results were verified through secondary biological assays and expression data were analyzed by a novel application of the Gene Set Enrichment Analysis (GSEA) method. CONCLUSIONS/SIGNIFICANCE: We screened 10,000 different cDNAs and identified a number of genes and pathways that have previously not been implicated in SREBP control and cellular cholesterol homeostasis. These findings further our understanding of lipid biology and should lead to new insights into lipid associated disorders.

Genome-scale functional profiling of the mammalian AP-1 signaling pathway.

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.