Showing papers in "Molecular and Cellular Biology in 2012"

Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks.

Abstract: Living cells are adaptive self-sustaining systems. They strictly depend on the sufficient supply of oxygen, energy, and nutrients from the outside in order to sustain their internal organization. However, as autonomous entities they are able to monitor and appropriately adapt to any critical fluctuation in their environment. In the case of insufficient external nutrient supply or augmented energy demands, cells start to extensively digest their own interior. This process, known as macroautophagy, comprises the transport of cytosolic portions and entire organelles to the lysosomal compartment via specific double-membrane vesicles, called autophagosomes. Although extensively upregulated under nutrient restriction, a low level of basal autophagy is likewise crucial in order to sustain the cellular homeostasis. On the other hand, cells have to avoid excessive and enduring self-digestion. The delicate balance between external energy and nutrient supply and internal production and consumption is a demanding task. The complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible posttranslational modifications such as phosphorylation. This review focuses on the serine/threonine protein kinases AMP-activated protein kinase, mammalian target of rapamycin (mTOR), and unc-51-like kinase 1/2 (Ulk1/2), three interconnected major junctions within the autophagy regulating signaling network.

Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle.

Abstract: Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

Abstract: The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Occludin: one protein, many forms.

Abstract: Intercellular tight junctions (TJs) exhibit a complex molecular architecture involving the regulated cointeraction of cytoplasmic adaptor proteins (e.g., zonula occludens) and integral membrane linker proteins (e.g., occludin and claudins). They provide structural integrity to epithelial and endothelial tissues and create highly polarized barriers essential to homeostatic maintenance within vertebrate physiological systems, while their dysregulation is an established pathophysiological hallmark of many diseases (e.g., cancer, stroke, and inflammatory lung disease). The junctional complex itself is a highly dynamic signaling entity wherein participant proteins constantly undergo a blend of regulatory modifications in response to diverse physiological and pathological cues, ultimately diversifying the overall adhesive properties of the TJ. Occludin, a 65-kDa tetraspan integral membrane protein, contributes to TJ stabilization and optimal barrier function. This paper reviews our current knowledge of how tissue occludin is specifically modified at the posttranscriptional and posttranslational levels in diverse circumstances, with associated consequences for TJ dynamics and epithelial/endothelial homeostasis. Mechanistic concepts such as splice variance and alternate promoter usage, proteolysis, phosphorylation, dimerization, and ubiquitination are comprehensively examined, and possible avenues for future investigation highlighted.

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Abstract: Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.

Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells.

Abstract: Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.

A Critical Role for the Wnt Effector Tcf4 in Adult Intestinal Homeostatic Self-Renewal

Abstract: Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1-/-, Tcf3(flox), and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.

Retinol-Binding Protein 4 Inhibits Insulin Signaling in Adipocytes by Inducing Proinflammatory Cytokines in Macrophages through a c-Jun N-Terminal Kinase- and Toll-Like Receptor 4-Dependent and Retinol-Independent Mechanism

Abstract: Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.

MicroRNA-200c represses migration and invasion of breast cancer cells by targeting actin-regulatory proteins FHOD1 and PPM1F.

Abstract: MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor β (TGF-β)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.

Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor α in mouse liver reveals extensive sharing of binding sites.

Abstract: The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A

Abstract: Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

A novel role for copper in Ras/mitogen-activated protein kinase signaling.

Abstract: Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.

Peyer's Patch M Cells Derived from Lgr5+ Stem Cells Require SpiB and Are Induced by RankL in Cultured “Miniguts”

Abstract: Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB(-/-) mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid ("minigut") cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.

Dopamine-regulated microRNA MiR-181a controls GluA2 surface expression in hippocampal neurons.

Abstract: The dynamic expression of AMPA-type glutamate receptors (AMPA-R) at synapses is a key determinant of synaptic plasticity, including neuroadaptations to drugs of abuse. Recently, microRNAs (miRNAs) have emerged as important posttranscriptional regulators of synaptic plasticity, but whether they target glutamate receptors to mediate this effect is not known. Here we used microarray screening to identify miRNAs that regulate synaptic plasticity within the nucleus accumbens, a brain region critical to forming drug-seeking habits. One of the miRNAs that showed a robust enrichment at medium spiny neuron synapses was miR-181a. Using bioinformatics tools, we detected a highly conserved miR-181a binding site within the mRNA encoding the GluA2 subunit of AMPA-Rs. Overexpression and knockdown of miR-181a in primary neurons demonstrated that this miRNA is a negative posttranscriptional regulator of GluA2 expression. Additionally, miR-181a overexpression reduced GluA2 surface expression, spine formation, and miniature excitatory postsynaptic current (mEPSC) frequency in hippocampal neurons, suggesting that miR-181a could regulate synaptic function. Moreover, miR-181a expression was induced by dopamine signaling in primary neurons, as well as by cocaine and amphetamines, in a mouse model of chronic drug treatment. Taken together, our results identify miR-181a as a key regulator of mammalian AMPA-type glutamate receptors, with potential implications for the regulation of drug-induced synaptic plasticity.

BNip3 regulates mitochondrial function and lipid metabolism in the liver.

Abstract: BNip3 localizes to the outer mitochondrial membrane, where it functions in mitophagy and mitochondrial dynamics. While the BNip3 protein is constitutively expressed in adult liver from fed mice, we have shown that its expression is superinduced by fasting of mice, consistent with a role in responses to nutrient deprivation. Loss of BNip3 resulted in increased lipid synthesis in the liver that was associated with elevated ATP levels, reduced AMP-regulated kinase (AMPK) activity, and increased expression of lipogenic enzymes. Conversely, there was reduced β-oxidation of fatty acids in BNip3 null liver and also defective glucose output under fasting conditions. These metabolic defects in BNip3 null liver were linked to increased mitochondrial mass and increased hepatocellular respiration in the presence of glucose. However, despite elevated mitochondrial mass, an increased proportion of mitochondria exhibited loss of mitochondrial membrane potential, abnormal structure, and reduced oxygen consumption. Elevated reactive oxygen species, inflammation, and features of steatohepatitis were also observed in the livers of BNip3 null mice. These results identify a role for BNip3 in limiting mitochondrial mass and maintaining mitochondrial integrity in the liver that has consequences for lipid metabolism and disease.

Glucose Oxidation Modulates Anoikis and Tumor Metastasis

Abstract: Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.

LGR5 Interacts and Cointernalizes with Wnt Receptors To Modulate Wnt/β-Catenin Signaling

Abstract: LGR5, a seven-transmembrane domain receptor of the rhodopsin family, is a Wnt target gene and a bona fide marker of adult stem cells in the gastrointestinal tract and hair follicle bulge. Recently, we and others demonstrated that LGR5 and its homologues function as receptors of the R-spondin family of stem cell factors to potentiate Wnt/β-catenin signaling. However, the mechanism of how LGR5 enhances the signaling output remains unclear. Here we report that following costimulation with the ligands R-spondin1 and Wnt3a, LGR5 interacts and forms a supercomplex with the Wnt coreceptors LRP6 and Fzd5 which is rapidly internalized and then degraded. Internalization of LGR5 is mediated through a dynamin- and clathrin-dependent pathway. Inhibition of this endocytic process has no effect on LGR5 signaling. Deletion of the C-terminal tail of LGR5 maintains its ability to interact with LRP6, yet this LGR5 mutant exhibits increased signaling activity and a decreased rate of endocytosis in response to R-spondin1 compared to the wild-type receptor. This study provides direct evidence that LGR5 becomes part of the Wnt signaling complex at the membrane level to enhance Wnt/β-catenin signaling. However, internalization of LGR5 does not appear to be essential for potentiating the canonical Wnt signaling pathway.

Rcf1 and Rcf2, members of the hypoxia-induced gene 1 protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex.

Abstract: We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.

Sam50 Functions in Mitochondrial Intermembrane Space Bridging and Biogenesis of Respiratory Complexes

Abstract: Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of β-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).

Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers.

Abstract: Autophagy is an evolutionarily conserved degradation pathway characterized by dynamic rearrangement of membranes that sequester cytoplasm, protein aggregates, organelles, and pathogens for delivery to the vacuole and lysosome, respectively. The ability of autophagosomal membranes to act selectively toward specific cargo is dependent on the small ubiquitin-like modifier ATG8/LC3 and the LC3-interacting region (LIR) present in autophagy receptors. Here, we describe a comprehensive protein-protein interaction analysis of TBC (Tre2, Bub2, and Cdc16) domain-containing Rab GTPase-activating proteins (GAPs) as potential autophagy adaptors. We identified 14 TBC domain-containing Rab GAPs that bind directly to ATG8 modifiers and that colocalize with LC3-positive autophagy membranes in cells. Intriguingly, one of our screening hits, TBC1D5, contains two LIR motifs. The N-terminal LIR was critical for interaction with the retromer complex and transport of cargo. Direct binding of the retromer component VPS29 to TBC1D5 could be titrated out by LC3, indicating a molecular switch between endosomes and autophagy. Moreover, TBC1D5 could bridge the endosome and autophagosome via its C-terminal LIR motif. During starvation-induced autophagy, TBC1D5 was relocalized from endosomal localization to the LC3-positive autophagosomes. We propose that LC3-interacting Rab GAPs are implicated in the reprogramming of the endocytic trafficking events under starvation-induced autophagy.

PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function

Abstract: PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.

SIRT3 functions in the nucleus in the control of stress-related gene expression.

Abstract: SIRT3 is a member of the Sir2 family of NAD+-dependent protein deacetylases that promotes longevity in many organisms. The processed short form of SIRT3 is a well-established mitochondrial protein whose deacetylase activity regulates various metabolic processes. However, the presence of full-length (FL) SIRT3 in the nucleus and its functional importance remain controversial. Our previous studies demonstrated that nuclear FL SIRT3 functions as a histone deacetylase and is transcriptionally repressive when artificially recruited to a reporter gene. Here, we report that nuclear FL SIRT3 is subjected to rapid degradation under conditions of cellular stress, including oxidative stress and UV irradiation, whereas the mitochondrial processed form is unaffected. FL SIRT3 degradation is mediated by the ubiquitin-proteasome pathway, at least partially through the ubiquitin protein ligase (E3) activity of SKP2. Finally, we show by chromatin immunoprecipitation that some target genes of nuclear SIRT3 are derepressed upon degradation of SIRT3 caused by stress stimuli. Thus, SIRT3 exhibits a previously unappreciated role in the nucleus, modulating the expression of some stress-related and nuclear-encoded mitochondrial genes.

The Saccharomyces cerevisiae Chromatin Remodeler Fun30 Regulates DNA End Resection and Checkpoint Deactivation

Abstract: Fun30 is a Swi2/Snf2 homolog in budding yeast that has been shown to remodel chromatin both in vitro and in vivo. We report that Fun30 plays a key role in homologous recombination, by facilitating 5'-to-3' resection of double-strand break (DSB) ends, apparently by facilitating exonuclease digestion of nucleosome-bound DNA adjacent to the DSB. Fun30 is recruited to an HO endonuclease-induced DSB and acts in both the Exo1-dependent and Sgs1-dependent resection pathways. Deletion of FUN30 slows the rate of 5'-to-3' resection from 4 kb/h to about 1.2 kb/h. We also found that the resection rate is reduced by DNA damage-induced phosphorylation of histone H2A-S129 (γ-H2AX) and that Fun30 interacts preferentially with nucleosomes in which H2A-S129 is not phosphorylated. Fun30 is not required for later steps in homologous recombination. Like its homolog Rdh54/Tid1, Fun30 is required to allow the adaptation of DNA damage checkpoint-arrested cells with an unrepaired DSB to resume cell cycle progression.

Integration of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival.

Abstract: The NKX3-1 gene is a homeobox gene required for prostate tumor progression, but how it functions is unclear. Here, using chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) we showed that NKX3-1 colocalizes with the androgen receptor (AR) across the prostate cancer genome. We uncovered two distinct mechanisms by which NKX3-1 controls the AR transcriptional network in prostate cancer. First, NKX3-1 and AR directly regulate each other in a feed-forward regulatory loop. Second, NKX3-1 collaborates with AR and FoxA1 to mediate genes in advanced and recurrent prostate carcinoma. NKX3-1- and AR-coregulated genes include those found in the “protein trafficking” process, which integrates oncogenic signaling pathways. Moreover, we demonstrate that NKX3-1, AR, and FoxA1 promote prostate cancer cell survival by directly upregulating RAB3B, a member of the RAB GTPase family. Finally, we show that RAB3B is overexpressed in prostate cancer patients, suggesting that RAB3B together with AR, FoxA1, and NKX3-1 are important regulators of prostate cancer progression. Collectively, our work highlights a novel hierarchical transcriptional regulatory network between NKX3-1, AR, and the RAB GTPase signaling pathway that is critical for the genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

The super elongation complex family of RNA polymerase II elongation factors: Gene target specificity and transcriptional output

Abstract: The elongation stage of transcription is highly regulated in metazoans. We previously purified the AFF1- and AFF4-containing super elongation complex (SEC) as a major regulator of development and cancer pathogenesis. Here, we report the biochemical isolation of SEC-like 2 (SEC-L2) and SEC-like 3 (SEC-L3) containing AFF2 and AFF3 in association with P-TEFb, ENL/MLLT1, and AF9/MLLT3. The SEC family members demonstrate high levels of polymerase II (Pol II) C-terminal domain kinase activity; however, only SEC is required for the proper induction of the HSP70 gene upon stress. Genome-wide mRNA-Seq analyses demonstrated that SEC-L2 and SEC-L3 control the expression of different subsets of genes, while AFF4/SEC plays a more dominant role in rapid transcriptional induction in cells. MYC is one of the direct targets of AFF4/SEC, and SEC recruitment to the MYC gene regulates its expression in different cancer cells, including those in acute myeloid or lymphoid leukemia. These findings suggest that AFF4/SEC could be a potential therapeutic target for the treatment of leukemia or other cancers associated with MYC overexpression.

A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development.

Abstract: The binding of the eukaryotic initiation factor 4E (eIF4E) to the mRNA 5′ cap structure is a rate-limiting step in mRNA translation initiation. eIF4E promotes ribosome recruitment to the mRNA. In Drosophila, the eIF4E homologous protein (d4EHP) forms a complex with binding partners to suppress the translation of distinct mRNAs by competing with eIF4E for binding the 5′ cap structure. This repression mechanism is essential for the asymmetric distribution of proteins and normal embryonic development in Drosophila. In contrast, the physiological role of the mammalian 4EHP (m4EHP) was not known. In this study, we have identified the Grb10-interacting GYF protein 2 (GIGYF2) and the zinc finger protein 598 (ZNF598) as components of the m4EHP complex. GIGYF2 directly interacts with m4EHP, and this interaction is required for stabilization of both proteins. Disruption of the m4EHP-GIGYF2 complex leads to increased translation and perinatal lethality in mice. We propose a model by which the m4EHP-GIGYF2 complex represses translation of a subset of mRNAs during embryonic development, as was previously reported for d4EHP.

Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein.

Abstract: Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

Critical and Reciprocal Regulation of KLF4 and SLUG in Transforming Growth Factor β-Initiated Prostate Cancer Epithelial-Mesenchymal Transition

Abstract: Epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of transforming growth factor β (TGFβ)-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, as demonstrated by the inhibition of EMT following Slug depletion. In contrast, SNAIL depletion was significantly less rate limiting. TGFβ-stimulated KLF4 degradation is required for SLUG induction. Expression of a degradation-resistant KLF4 mutant inhibited EMT, and furthermore, depletion of Klf4 was sufficient to initiate SLUG-dependent EMT. We show that KLF4 and another epithelial determinant, FOXA1, are direct transcriptional inhibitors of SLUG expression in mouse and human prostate cancer cells. Furthermore, self-reinforcing regulatory loops for SLUG-KLF4 and SLUG-FOXA1 lead to SLUG-dependent binding of polycomb repressive complexes to the Klf4 and Foxa1 promoters, silencing transcription and consolidating mesenchymal commitment. Analysis of tissue arrays demonstrated decreased KLF4 and increased SLUG expression in advanced-stage primary prostate cancer, substantiating the involvement of the EMT signaling events described in model systems.

The E3 ubiquitin ligase TRAF6 intercedes in starvation-induced skeletal muscle atrophy through multiple mechanisms.

Abstract: Starvation, like many other catabolic conditions, induces loss of skeletal muscle mass by promoting fiber atrophy. In addition to the canonical processes, the starvation-induced response employs many distinct pathways that make it a unique atrophic program. However, in the multiplex of the underlying mechanisms, several components of starvation-induced atrophy have yet to be fully understood and their roles and interplay remain to be elucidated. Here we unveiled the role of tumor necrosis factor receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase and adaptor protein, in starvation-induced muscle atrophy. Targeted ablation of TRAF6 suppresses the expression of key regulators of atrophy, including MAFBx, MuRF1, p62, LC3B, Beclin1, Atg12, and Fn14. Ablation of TRAF6 also improved the phosphorylation of Akt and FoxO3a and inhibited the activation of 5' AMP-activated protein kinase in skeletal muscle in response to starvation. In addition, our study provides the first evidence of the involvement of endoplasmic reticulum stress and unfolding protein response pathways in starvation-induced muscle atrophy and its regulation through TRAF6. Finally, our results also identify lysine 63-linked autoubiquitination of TRAF6 as a process essential for its regulatory role in starvation-induced muscle atrophy.

Berardinelli-Seip Congenital Lipodystrophy 2/Seipin Is a Cell-Autonomous Regulator of Lipolysis Essential for Adipocyte Differentiation

Abstract: Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2(-/-) mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance, and hepatic steatosis. In vitro differentiation of both Bscl2(-/-) murine embryonic fibroblasts (MEFs) and stromal vascular cells (SVCs) reveals normal early-phase adipocyte differentiation but a striking failure in terminal differentiation due to unbridled cyclic AMP (cAMP)-dependent protein kinase A (PKA)-activated lipolysis, which leads to loss of lipid droplets and silencing of the expression of adipose tissue-specific transcription factors. Importantly, such defects in differentiation can be largely rescued by inhibitors of lipolysis but not by a gamma peroxisome proliferator-activated receptor (PPARγ) agonist. The residual epididymal WAT (EWAT) in Bscl2(-/-) mice displays enhanced lipolysis. It also assumes a "brown-like" phenotype with marked upregulation of UCP1 and other brown adipose tissue-specific markers. Together with decreased Pref1 but increased C/EBPβ levels, these changes highlight a possible increase in cAMP signaling that impairs terminal adipocyte differentiation in the EWAT of Bscl2(-/-) mice. Our study underscores the fundamental role of regulated cAMP/PKA-mediated lipolysis in adipose differentiation and identifies Bscl2 as a novel cell-autonomous determinant of activated lipolysis essential for terminal adipocyte differentiation.