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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in ...tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.
High-content screening is transforming drug discovery by enabling simultaneous measurement of multiple features of cellular phenotype that are relevant to therapeutic and toxic activities of ...compounds. High-content screening studies typically generate immense datasets of image-based phenotypic information, and how best to mine relevant phenotypic data is an unsolved challenge. Here, we introduce factor analysis as a data-driven tool for defining cell phenotypes and profiling compound activities. This method allows a large data reduction while retaining relevant information, and the data-derived factors used to quantify phenotype have discernable biological meaning. We used factor analysis of cells stained with fluorescent markers of cell cycle state to profile a compound library and cluster the hits into seven phenotypic categories. We then compared phenotypic profiles, chemical similarity and predicted protein binding activities of active compounds. By integrating these different descriptors of measured and potential biological activity, we can effectively draw mechanism-of-action inferences.
PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To ...identify upstream regulators of PGC-1α gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1α promoter. A number of activators of PGC-1α transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB. The other two members of the TORC family, TORC2 and TORC3, also strongly activated PGC-1α transcription. TORCs dramatically induced PGC-1α gene transcription through CREB. Forced expression of TORCs in primary muscle cells induced the endogenous mRNA of PGC-1α and its downstream target genes in the mitochondrial respiratory chain and TCA cycle. Importantly, these changes in gene expression resulted in increased mitochondrial oxidative capacity measured by cellular respiration and fatty acid oxidation. Finally, we demonstrated that the action of TORCs in promoting mitochondrial gene expression and function requires PGC-1α. Previous studies had indicated that TORCs function as a calcium- and cAMP-sensitive coincidence detector and mediate individual and synergistic effects of these two pathways. Our results, together with previous findings, strongly suggest that TORCs play a key role in linking these external signals to the transcriptional program of adaptive mitochondrial biogenesis by activating PGC-1α gene transcription.
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.
The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation 1–4. The ...TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown 5, 6. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, was mediated by calcineurin, and TORC1 was shown to be directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 was sufficient to activate CRE-dependent transcription. Drosophila TORC was also shown to translocate in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During long-term treatment of various malignant or viral diseases with IFN-alpha up to 20% of patients develop anti-IFN-alpha antibodies for as yet unknown reasons.
To address this issue, a mouse ...model using Balb/C mice was established and the relevance of several potentially anti-IFN-alpha antibodies inducing factors was studied.
The model revealed that both a higher frequency of injections and a higher dosage of IFN-alpha were more immunogenic and that the route of administration affected the antibody response to IFN-alpha. The intrinsic immunostimulatory activity of IFN-alpha itself also enhanced the immune response. IFN-alpha protein aggregates (IFN-alpha-IFN-alpha and human serum albumin (HSA)-IFN-alpha aggregates), which were recently identified in all marketed IFN-alpha products, were significantly more immunogenic than IFN-alpha monomers. These aggregates broke the tolerance against human IFN-alpha monomers in human IFN-alpha transgenic mice.
Based on these animal studies it is proposed that the immune response to IFN-alpha in humans is most probably elicited by a combination of several factors among which IFN-alpha protein aggregates seem to play a key role.
PDF
