Small-Molecule Reactivation of Mutant p53 to Wild-Type-like p53 through the p53-Hsp40 Regulatory Axis.

TP53 is the most frequently mutated gene in human cancer, and small-molecule reactivation of mutant p53 function represents an important anticancer strategy. A cell-based, high-throughput small-molecule screen identified chetomin (CTM) as a mutant p53 R175H reactivator. CTM enabled p53 to transactivate target genes, restored MDM2 negative regulation, and selectively inhibited the growth of cancer cells harboring mutant p53 R175H in vitro and in vivo. We found that CTM binds to Hsp40 and increases the binding capacity of Hsp40 to the p53 R175H mutant protein, causing a potential conformational change to a wild-type-like p53. Thus, CTM acts as a specific reactivator of the p53 R175H mutant form through Hsp40. These results provide new insights into the mechanism of reactivation of this specific p53 mutant.

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53.

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway.

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.

Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors.

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a ß-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

CDIP1-BAP31 complex transduces apoptotic signals from endoplasmic reticulum to mitochondria under endoplasmic reticulum stress.

Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress.

The Ser/Thr Rho kinase 1 (ROCK1) is known to have major roles in a wide range of cellular activities, including those involved in tumour metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the phosphoinositide 3-kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy.

Deep sequencing analysis of phage libraries using Illumina platform.

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of phage clones and identify ligands previously lost in phage display screens.

GAMT joins the p53 network: branching into metabolism.

The p53 protein functions to prevent tumor development by restricting proliferation, motility and survival of abnormal or stressed cells. In addition to well-established roles, recent discoveries indicate a role for p53 in the regulation of pathways involved in energy metabolism. The metabolic functions of p53 can inhibit the shift to glycolysis that is characteristically seen in cancer cells, while favoring the energy production by mitochondrial oxidative phosphorylation. Identification of guanidinoacetate methyltransferase (GAMT) as a new p53 target connects p53 to creatine metabolism critical in the regulation of ATP homeostasis. The involvement of GAMT in both genotoxic and metabolic stressinduced apoptosis, as well as the requirement of p53-dependent upregulation of GAMT in glucose starvation-mediated fatty acid oxidation (FAO), demonstrate a further role of p53 in coordinating stress response with changes in cellular metabolism. Such activities of p53 would help to bring a better understanding of how cancer cells acquire unique metabolic features to maintain their own survival and proliferation, and might provide interesting clues toward the development of novel therapies.

Loss of Stearoyl-CoA desaturase-1 attenuates adipocyte inflammation: effects of adipocyte-derived oleate.

BACKGROUND AND PURPOSE: Adipose inflammation is crucial to the pathogenesis of metabolic disorders. This study aimed at identify the effects of stearoyl-CoA desaturase-1 (SCD1) on the inflammatory response of a paracrine network involving adipocytes, macrophages, and endothelial cells. METHODS AND RESULTS: Loss of SCD1 in both genetic (Agouti) and diet-induced obesity (high-fat diet) mouse models prevented inflammation in white adipose tissue and improved its basal insulin signaling. In SCD1-deficient mice, white adipose tissue exhibited lower inflammation, with a reduced response to lipopolysaccharide in isolated adipocytes, but not in peritoneal macrophages. Mimicking the in vivo paracrine regulation of white adipose tissue inflammation, SCD1-deficient adipocyte-conditioned medium attenuated the induction of tumor necrosis factor (TNF) alpha/interleukin 1beta gene expression in RAW264.7 macrophages and reduced the adhesion response in endothelial cells. We further demonstrated that the adipocyte-derived oleate (18:1n9), but not palmitoleate (16:1n7), mediated the inflammation in macrophages and adhesion responses in endothelial cells. CONCLUSIONS: Loss of SCD1 attenuates adipocyte inflammation and its paracrine regulation of inflammation in macrophages and endothelial cells. The reduced oleate level is linked to the inflammation-modulating effects of SCD1 deficiency.

GAMT, a p53-inducible modulator of apoptosis, is critical for the adaptive response to nutrient stress.

The p53 tumor suppressor protein has a well-established role in cell-fate decision-making processes. However, recent discoveries indicate that p53 has a non-tumor-suppressive role. Here we identify guanidinoacetate methyltransferase (GAMT), an enzyme involved in creatine synthesis, as a p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53-dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53-->GAMT upregulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP-generating energy source. These results highlight that p53-dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. The p53-->GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism.

Skin-specific deletion of stearoyl-CoA desaturase-1 alters skin lipid composition and protects mice from high fat diet-induced obesity.

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids and is an important regulator of whole body energy homeostasis. Severe cutaneous changes in mice globally deficient in SCD1 also indicate a role for SCD1 in maintaining skin lipids. We have generated mice with a skin-specific deletion of SCD1 (SKO) and report here that SKO mice display marked sebaceous gland hypoplasia and depletion of sebaceous lipids. In addition, SKO mice have significantly increased energy expenditure and are protected from high fat diet-induced obesity, thereby recapitulating the hypermetabolic phenotype of global SCD1 deficiency. Genes of fat oxidation, lipolysis, and thermogenesis, including uncoupling proteins and peroxisome proliferator-activated receptor-gamma co-activator-1alpha, are up-regulated in peripheral tissues of SKO mice. However, unlike mice globally deficient in SCD1, SKO mice have an intact hepatic lipogenic response to acute high carbohydrate feeding. Despite increased basal thermogenesis, SKO mice display severe cold intolerance because of rapid depletion of fuel substrates, including hepatic glycogen, to maintain core body temperature. These data collectively indicate that SKO mice have increased cold perception because of loss of insulating factors in the skin. This results in up-regulation of thermogenic processes for temperature maintenance at the expense of fuel economy, illustrating cross-talk between the skin and peripheral tissues in maintaining energy homeostasis.

Stearoyl-CoA desaturase-1 deficiency attenuates obesity and insulin resistance in leptin-resistant obese mice.

Obesity and adiposity greatly increase the risk for secondary conditions such as insulin resistance. Mice deficient in the enzyme stearoyl-CoA desaturase-1 (SCD1) are lean and protected from diet-induced obesity and insulin resistance. In order to determine the effect of SCD1 deficiency on various mouse models of obesity, we introduced a global deletion of the Scd1 gene into leptin-deficient ob/ob mice, leptin-resistant Agouti (A(y)/a) mice, and high-fat diet-fed obese (DIO) mice. SCD1 deficiency lowered body weight, adiposity, hepatic lipid accumulation, and hepatic lipogenic gene expression in all three mouse models. However, glucose tolerance, insulin, and leptin sensitivity were improved by SCD1 deficiency only in A(y)/a and DIO mice, but not ob/ob mice. These data uncouple the effects of SCD1 deficiency on weight loss from those on insulin sensitivity and suggest a beneficial effect of SCD1 inhibition on insulin sensitivity in obese mice that express a functional leptin gene.

Hepatic stearoyl-CoA desaturase-1 deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis.

Stearoyl-CoA desaturase-1 (SCD1), a critical regulator of energy metabolism, catalyzes the synthesis of monounsaturated fats. To understand the tissue-specific role of SCD1 in energy homeostasis, we used Cre-lox technology to generate mice with a liver-specific knockout of Scd1 (LKO). LKO mice were protected from high-carbohydrate, but not high-fat (HF), diet-induced adiposity and hepatic steatosis. Additionally, on a high-sucrose, very low-fat (HSVLF) diet, lipogenesis and levels of nuclear SREBP-1 and ChREBP were significantly decreased in the livers of LKO relative to Scd1(lox/lox) (Lox) mice. HSVLF feeding in LKO mice caused hypoglycemia and hepatic carbohydrate reduction due to an impairment of gluconeogenesis. Oleate, but not stearate, supplementation normalized adiposity, gluconeogenesis, triglyceride secretion, and hepatic lipogenesis of LKO mice. These results indicate that hepatic SCD1 expression (and thus, oleate) is required for carbohydrate-induced adiposity, but SCD1 inhibition in extrahepatic tissues is required to protect mice from HF-induced obesity and insulin resistance.

Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation.

Stearoyl-coenzyme A desaturase (SCD) is the rate-limiting enzyme necessary for the biosynthesis of monounsaturated fatty acids. In this study, we investigated the regulation of mouse SCD1 by liver X receptor (LXR) and its role in plasma lipoprotein metabolism upon LXR activation. In vivo, the SCD1 gene remained induced upon LXR activation in the absence of sterol regulatory element-binding protein 1c (SREBP-1c), a known transcriptional regulator of SCD1. Serial deletion and point mutation analyses in reporter gene assays, as well as a gel mobility shift assay, identified an LXR response element in the mouse SCD1 promoter. In addition, SCD1 deficiency prevented the hypertriglyceridemic effect and reduced hepatic triglyceride accumulation associated with LXR activation despite induced hepatic expression of SREBP-1c protein and several SREBP1c and LXR target genes involved in lipoprotein metabolism. Unlike wild-type mice, SCD1-deficient mice failed to elevate the hepatic triglyceride monounsaturated acid (MUFA)/saturated fatty acid (SFA) ratio despite induction of the SCD2 gene. Together, these findings suggest that SCD1 plays a pivotal role in the regulation of hepatic and plasma triglyceride accumulation, possibly by modulating the MUFA-to-SFA ratio. In addition, SCD1 deficiency also increased plasma high-density lipoprotein cholesterol levels induced by LXR activation.

Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis.

Stearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) protein that catalyzes the Delta9-cis desaturation of saturated fatty acids. Mice with targeted disruption in SCD1 (Scd1(-/-)) have significant reduction in the tissue content of triglycerides, suggesting that monounsaturated fatty acids endogenously synthesized by SCD1 are important for triglyceride synthesis. Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is the enzyme that catalyzes the final reaction in the synthesis of triglycerides. The lack of DGAT2, one of the two DGAT isoforms, results in almost a complete loss of tissue triglycerides. We hypothesize that SCD1 participates in triglyceride synthesis by providing a more accessible pool of monounsaturated fatty acids through substrate channeling. In this study, we test whether SCD1 is proximal to DGAT2 by colocalization study with confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer using HeLa cells as the model of study. All of the results suggest that SCD1 and DGAT2 are located very close to each other in the ER, which is a very important criterion for the channeling of substrate. By performing subcellular fractionation using mouse livers, we also show, for the first time, that SCD is present in the mitochondria-associated membrane.

Membrane topology of mouse stearoyl-CoA desaturase 1.

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.

Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver.

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.

Reduced adiposity and liver steatosis by stearoyl-CoA desaturase deficiency are independent of peroxisome proliferator-activated receptor-alpha.

Stearoyl-CoA desaturase catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids, which are required for normal rates of synthesis of triglycerides, cholesterol esters, and phospholipids. Mice with a targeted disruption of the stearoyl-CoA desaturase 1 (SCD1) isoform are protected against diet and leptin deficiency-induced adiposity, have increased energy expenditure, and have up-regulated expression of hepatic genes encoding enzymes of fatty acid beta-oxidation. Because peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key transcription factor that induces the transcription of fatty acid beta-oxidation and thermogenic genes, we hypothesized that the increased fatty acid oxidation observed in SCD1 deficiency is dependent on activation of the PPARalpha pathway. Here we show that mice nullizygous for SCD1 and PPARalpha are still protected against adiposity, have increased energy expenditure, and maintain high expression of PPARalpha target genes in the liver and brown adipose tissue. The SCD1 deficiency rescued hepatic steatosis of the PPARalpha(-/-) mice. The SCD1 mutation increased the phosphorylation of both AMP-activated protein kinase and acetyl-CoA carboxylase, thereby increasing CPT activity and stimulating the oxidation of liver palmitoyl-CoA in the PPARalpha null mice. The findings indicate that the reduced adiposity, reduced liver steatosis, increased energy expenditure, and increased expression of PPARalpha target genes associated with SCD1 deficiency are independent of activation of the PPARalpha pathway.

Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms.

Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1-/- mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c-/- mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis.