CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

PRAP1 is a novel lipid-binding protein that promotes lipid absorption by facilitating MTTP-mediated lipid transport.

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.

Fracture risks in patients with atopic dermatitis: A nationwide matched cohort study.

BACKGROUND: The risk of osteoporosis has been explored in atopic dermatitis (AD). The long-term risk of fractures in patients with AD and the effects of various AD treatments on bone health remain to be elucidated. OBJECTIVE: To evaluate the long-term risk of fractures in patients with AD. METHODS: This nationwide matched cohort study was conducted using the National Health Insurance Research Database of Taiwan for the period 1997 to 2013. A total of 36,855 patients with AD and 147,420 reference subjects without AD were identified. Demographic characteristics and comorbidities were compared, and cumulative incidence of fractures was evaluated. Adjusted hazard ratios for fracture risks of AD and various AD treatments were calculated using the Cox proportional hazards model. RESULTS: A total of 1518 patients (4.12%) in the AD cohort and 5579 patients (3.78%) in the reference cohort had fractures (P = .003). The mean ages were 22.6 years in both groups. The 16-year cumulative incidence of fractures in the AD cohort (8.043%) was significantly higher than that in the reference cohort (7.366%) (P = .002). Severe AD (adjusted hazard ratio [aHR], 1.31; 95% confidence interval [CI], 1.08-1.59) was independently associated with fractures. Other independent risk factors included exposure to topical (aHR, 1.21; 95% CI, 1.05-1.39) or systemic (≥10 mg/d; aHR, 1.62; 95% CI, 1.38-1.91) corticosteroids. Use of disease-modifying antirheumatic drugs (aHR, 0.71; 95% CI, 0.53-0.90) and phototherapy (aHR, 0.73; 95% CI, 0.56-0.95) was associated with a lower risk of fractures. The results were consistent across sensitivity analyses. CONCLUSION: Patients with AD have a higher incidence of fractures. Severe AD is independently associated with fractures.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet.

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Thymic epithelial ß-catenin is required for adult thymic homeostasis and function.

The role of ß-catenin in thymocyte development has been extensively studied, however, the function of ß-catenin in thymic epithelial cells (TECs) remains largely unclear. Here, we demonstrate a requirement for ß-catenin in keratin 5 (K5)-expressing TECs, which comprise the majority of medullary TECs (mTECs) and a progenitor subset for cortical TECs (cTECs) in the young adult thymus. We found that conditionally ablated ß-catenin in K5(+)-TECs and their progeny cells resulted in thymic atrophy. The composition of TECs was also aberrantly affected. Percentages of K5(hi)K8(+)-TECs, K5(+)K8(-)-TECs and UEA1(+)-mTECs were significantly decreased and the percentage of K5(lo)K8(+)-TECs and Ly51(+)-cTECs were increased in ß-catenin-deficient thymi compared with that in the control thymi. We also observed that ß-catenin-deficient TEC lineage could give rise to K8(+)-cTECs more efficiently than wild-type TECs using lineage-tracing approach. Importantly, the expression levels of several transcription factors (p63, FoxN1 and Aire), which are essential for TEC differentiation, were altered in ß-catenin-deficient thymi. Under the aberrant differentiation of TECs, development of all thymocytes in ß-catenin-deficient thymi was impaired. Interleukin-7 (IL-7) and chemokines (Ccl19, Ccl25 and Cxcl12) levels were also downregulated in the thymic stromal cells in the mutants. Finally, introducing a BCL2 transgene in lymphoid lineages, which has been shown to rescue IL-7-deficient thymopoiesis, partially rescued the thymic atrophy and thymocyte development defects caused by induced ablation of ß-catenin in K5(+)-TECs. Collectively, these findings suggest that ß-catenin is required for the differentiation of TECs, thereby contributing to thymocyte development in the postnatal thymus.

Mice with alopecia, osteoporosis, and systemic amyloidosis due to mutation in Zdhhc13, a gene coding for palmitoyl acyltransferase.

Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein-protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea-mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.

International Staging System predicts prognosis of Chinese patients with multiple myeloma across different calendar periods with application of novel agents.

The applicability of the International Staging System (ISS) for Chinese patients with multiple myeloma (MM) has not been demonstrated, especially with respect to treatments with novel agents. Newly diagnosed MM patients at Taipei Veterans General Hospital were enrolled between 1996 and 2007. Data regarding clinical features, laboratory tests, and outcome at last follow-up were collected. A total of 389 MM patients (71% male) were enrolled, with median age of 71 years. At diagnosis, 72.7% had Durie-Salmon (DS) stage III disease, 56.2% had ISS stage III disease, and 34% had serum creatinine ≧2.0 mg/dL. Compared with patients diagnosed in the first calendar period 1996-2001, the patients of the second calendar period 2002-2007 were older and more of these patients had received novel agents, especially thalidomide. The median overall survival period was 20.5 months, with a significant increase of patients in the second calendar period (15.3 and 28.2 months, respectively; P = 0.002), especially for those with ISS stages I and II. In the Cox proportion model, elevated serum ß(2) microglobulin at diagnosis (≧3.5 mg/L), old age (≧65 years), and impaired renal function were found to be independently associated with poor survival. Over the entire period, the ISS was found to be effective in providing an accurate prognosis with respect to different ages and calendar periods. This is the first study to show the applicability of ISS for Chinese patients with MM, especially for those who had received thalidomide.

Synergism between p53 and Mcl-1 in protecting from hepatic injury, fibrosis and cancer.

BACKGROUND & AIMS: Mcl-1-deficient hepatocytes are prone to undergo apoptosis. The tumor suppressor protein p53 plays an important role in apoptosis control as well as other cellular responses. This study was initially aimed to examine whether p53 was involved in Mcl-1 deficiency-induced apoptosis of hepatocytes. METHODS: Hepatocyte-specific Mcl-1 knockout (Alb-Mcl-1(-/-)) mice and Alb-Mcl-1(-/-) mice in wild-type or p53-deficient background were generated and characterized. RESULTS: Alb-Mcl-1(-/-) mice were viable, but their liver cells were prone to undergo apoptosis and manifested a slightly elevated level of p53. To examine the role of p53 in Alb-Mcl-1(-/-) livers, Alb-Mcl-1(-/-) mice without p53 (DKO mice) were characterized. Unexpectedly, although p53-deficient mice appeared to be developmentally normal, DKO mice were highly susceptible to neonatal death (∼60%). Further analysis revealed that such an early lethality was likely due to hepatic failure caused by a marked reduction of fully-differentiated hepatocytes at the perinatal/neonatal stage. Moreover, those DKO mice that did survive to adulthood manifested more severe liver damage than Alb-Mcl-1(-/-) mice, suggesting that p53 was activated in Alb-Mcl-1(-/-) livers to promote cell survival. Microarray followed by quantitative PCR analysis suggested that p21(Waf1/Cip1), one p53 target gene with apoptosis-inhibitory function, is likely involved in the protective role of p53 in Alb-Mcl-1(-/-) livers. Moreover, we demonstrated that loss of p53 promoted liver fibrosis and tumor development in Alb-Mcl-1(-/-) mice. CONCLUSIONS: This study revealed an unexpected synergism between Mcl-1 and p53 in protecting from hepatic injury, fibrosis, and cancer.

Promoter knock-in mutations reveal a role of Mcl-1 in thymocyte-positive selection and tissue or cell lineage-specific regulation of Mcl-1 expression.

We previously demonstrated that IL-3 stimulates transcription of the antiapoptotic gene mcl-1 via two promoter elements designated as the SIE and CRE-2 sites. To further study the functional role of these two DNA elements, mutant mice with targeted mutations of both SIE and CRE-2 sites (SC mutants) were generated. Homozygous SC mutants manifested a markedly reduced level of Mcl-1 in thymus but not in other major organs such as spleen, liver, lung, or heart. Reduced expression of Mcl-1 in SC mutant thymus resulted in attenuated positive selection of double-positive thymocytes into both CD4 and CD8 lineages, a result likely due to reduced survival of SC mutant double-positive thymocytes that were supposed to be positively selected. In contrast, in the peripheral lymphoid organs, only CD8(+) but not CD4(+) T cells were significantly reduced in homozygous SC mutant mice, a result consistent with a more dramatic decrease both of Mcl-1 expression and cell viability in mutant CD8(+) compared with mutant CD4(+) T cells. Impaired T cell development and peripheral CD8(+) lymphopenia in homozygous SC mutant mice were both cell autonomous and could be rescued by enforced expression of human Mcl-1. Together, the promoter-knock-in mouse model generated in this study not only revealed a role of Mcl-1 in thymocyte-positive selection, but also uncovered that Mcl-1 expression is regulated in a tissue or cell lineage-specific manner.

Critical roles of translationally controlled tumor protein in the homeostasis and TCR-mediated proliferation of peripheral T cells.

Translationally controlled tumor protein (TCTP) is expressed throughout T cell development and prominently induced following T cell activation. However, its function(s) during these processes is unclear. Here, we demonstrated that conditional deletion of TCTP before the beta selection checkpoint resulted into a partial block of thymocyte development at the double-negative (DN) 3 stage. Deletion of TCTP in the double-positive (DP) stage did not cause any significant phenotype in the thymus except a slight increase of mature CD8 single-positive (SP) thymocytes. In contrast to the very modest phenotype observed in the thymus, a significant reduction of mature T cells was observed in the peripheral lymphoid organs of these two conditional null TCTP mutant mice. Detailed analysis revealed that the latter phenotype (peripheral T cell lymphopenia) was largely due to a decreased viability of mature TCTP-deficient (TCTP(-/-)) T cells. Transgenic expression of the anti-apoptotic protein Bcl-2 rescued the partial block of early thymocyte development, but not peripheral T cell lymphopenia of T-lineage-specific TCTP(-/-) mice, suggesting that the signaling networks of TCTP in these two processes are not identical. Last, we demonstrated that TCTP(-/-) T cells manifested a significant defect in T cell Ag receptor (TCR)-mediated cell proliferation. Further analysis revealed that such defect was due to a marked delay in the initial cell-cycle entry of TCTP(-/-) T cells following TCR stimulation. Together, these results indicate that TCTP plays a very modest role in thymocyte development, but is critical for peripheral T cell maintenance and TCR-mediated cell proliferation.

Palmitoyl Acyltransferase Activity of ZDHHC13 Regulates Skin Barrier Development Partly by Controlling PADi3 and TGM1 Protein Stability.

Deficiency of the palmitoyl-acyl transferase ZDHHC13 compromises skin barrier permeability and renders mice susceptible to environmental bacterial infection and inflammatory dermatitis. It had been unclear how the lack of ZDHHC13 proteins resulted in cutaneous abnormalities. In this study, we first demonstrate that enzymatic palmitoylation activity, rather than protein scaffolding, by ZDHHC13 is essential for skin barrier integrity, showing that knock-in mice bearing an enzymatically dead DQ-to-AA ZDHHC13 mutation lost their hair after weaning cyclically, recapitulating knockout phenotypes of skin inflammation and dermatitis. To establish the ZDHHC13 substrates responsible for skin barrier development, we employed quantitative proteomic approaches to identify protein molecules whose palmitoylation is tightly controlled by ZDHHC13. We identified over 300 candidate proteins that could be classified into four biological categories: immunological disease, skin development and function, dermatological disease, and lipid metabolism. Palmitoylation of three of these candidates-loricrin, peptidyl arginine deiminase type III, and keratin fiber crosslinker transglutaminase 1-by ZDHHC13 was confirmed by biochemical assay. Palmitoylation was critical for in vivo protein stability of the latter two candidates. Our findings reveal the importance of protein palmitoylation in skin barrier development, partly by promoting envelope protein crosslinking and the filaggrin processing pathway.

CBAP modulates Akt-dependent TSC2 phosphorylation to promote Rheb-mTORC1 signaling and growth of T-cell acute lymphoblastic leukemia.

High-frequency relapse remains a clinical hurdle for complete remission of T-cell acute lymphoblastic leukemia (T-ALL) patients, with heterogeneous dysregulated signaling profiles-including of Raf-MEK-ERK and Akt-mTORC1-S6K signaling pathways-recently being implicated in disease outcomes. Here we report that GM-CSF/IL-3/IL-5 receptor common ß-chain-associated protein (CBAP) is highly expressed in human T-ALL cell lines and many primary tumor tissues and is required to bolster leukemia cell proliferation in tissue culture and for in vivo leukemogenesis in a xenograft mouse model. Downregulation of CBAP markedly restrains expansion of leukemia cells and alleviates disease aggravation of leukemic mice. Transcriptomic profiling and molecular biological analyses suggest that CBAP acts upstream of Ras and Rac1, and functions as a modulator of both Raf-MEK-ERK and Akt-mTORC1 signaling pathways to control leukemia cell growth. Specifically, CBAP facilitated Akt-dependent TSC2 phosphorylation in cell-based assays and in vitro analysis, decreased lysosomal localization of TSC2, and elevated Rheb-GTP loading and subsequent activation of mTORC1 signaling. Taken together, our findings reveal a novel oncogenic contribution of CBAP in T-ALL leukemic cells, in addition to its original pro-apoptotic function in cytokine-dependent cell lines and primary hematopoietic cells, by demonstrating its functional role in the regulation of Akt-TSC2-mTORC1 signaling for leukemia cell proliferation. Thus, CBAP represents a novel therapeutic target for many types of cancers and metabolic diseases linked to PI3K-Akt-mTORC1 signaling.

Identification of UAP1L1 as a critical factor for protein O-GlcNAcylation and cell proliferation in human hepatoma cells.

Aged hepatocyte-specific-Mcl-1 knockout (MKO-hep) mice are prone to develop liver tumors mimicking human hepatocellular carcinoma (HCC). Here we reported that a protein named UDP-N-acetylglucosamine pyrophosphorylase-1-like-1 (Uap1l1) is upregulated in the liver of young MKO-hep mice without any macroscopically detectable tumor nodules and is prominently expressed in the hepatic tumors developed in the aged MKO-hep mice. Intriguingly, human UAP1L1 is also significantly upregulated in a distinct subset of HCC tissues and patients with upregulated expression of UAP1L1 appeared to have poor prognosis. Overexpression of UAP1L1 significantly promoted, whereas UAP1L1 knockdown markedly reduced the proliferation of human hepatoma cells both in vitro and in vivo. UAP1L1 shows ~59% sequence identity to UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1), which is directly involved in the synthesis of the sugar donor (UDP-GlcNac) for N-acetylglucosamine modification (O-GlcNAcylation) of proteins. However, unlike UAP1, UAP1L1 harbors very limited UDP-GlcNAc synthesis activity. Moreover, although both UAP1 and UAP1L1 are required for O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation, they appear to function distinctly from each other. UAP1L1 directly interacts with OGT, but does not seem to be an OGT substrate. In addition, UAP1L1 alone is not sufficient to activate OGT activity in vitro, suggesting that UAP1L1 may function together with other proteins to modulate OGT activity in vivo. Lastly, UAP1L1 knockdown attenuated c-MYC O-GlcNAcylation and protein stability, and overexpression of c-MYC significantly rescued the proliferation defect of UAP1L1 knockdown HepG2 cells, suggesting that c-MYC is one downstream target of UAP1L1 that contributes to UAP1L1-mediated cell proliferation, at least in HepG2 cells.

Design and synthesis of potent dual inhibitors of JAK2 and HDAC based on fusing the pharmacophores of XL019 and vorinostat.

Specifically blocking more than one oncogenic pathway simultaneously in a cancer cell with a combination of different drugs is the mainstay of the majority of cancer treatments. Being able to do this via two targeted pathways without inducing side effects through a general mechanism, such as chemotherapy, could bring benefit to patients. In this work we describe a new dual inhibitor of the JAK-STAT and HDAC pathways through designing and developing two types of molecule based on the JAK2 selective inhibitor XL019 and the pan-HDAC inhibitor, vorinostat. Both series of compounds had examples with low nanomolar JAK2 and HDAC1/6 inhibition. In some cases good HDAC1 selectivity was achieved while retaining HDAC6 activity. The observed potency is explained through molecular docking studies of all three enzymes. One example, 69c had 16-25 fold selectivity against the three other JAK-family proteins JAK1, JAK3 and TYK2. A number of compounds had sub-micromolar potencies against a panel of 4 solid tumor cell lines and 4 hematological cell lines with the most potent compound, 45h, having a cellular IC50 of 70 nM against the multiple myeloma cell line KMS-12-BM. Evidence of both JAK and HDAC pathway inhibition is presented in Hela cells showing that both pathways are modulated. Evidence of apoptosis with two compounds in 4 sold tumor cell lines is also presented.

Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction.

JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.

Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver.

Palmitoyltransferase (PAT) catalyses protein S-palmitoylation which adds 16-carbon palmitate to specific cysteines and contributes to various biological functions. We previously reported that in mice, deficiency of Zdhhc13, a member of the PAT family, causes severe phenotypes including amyloidosis, alopecia, and osteoporosis. Here, we show that Zdhhc13 deficiency results in abnormal liver function, lipid abnormalities, and hypermetabolism. To elucidate the molecular mechanisms underlying these disease phenotypes, we applied a site-specific quantitative approach integrating an alkylating resin-assisted capture and mass spectrometry-based label-free strategy for studying the liver S-palmitoylome. We identified 2,190 S-palmitoylated peptides corresponding to 883 S-palmitoylated proteins. After normalization using the membrane proteome with TMT10-plex labelling, 400 (31%) of S-palmitoylation sites on 254 proteins were down-regulated in Zdhhc13-deficient mice, representing potential ZDHHC13 substrates. Among these, lipid metabolism and mitochondrial dysfunction proteins were overrepresented. MCAT and CTNND1 were confirmed to be specific ZDHHC13 substrates. Furthermore, we found impaired mitochondrial function in hepatocytes of Zdhhc13-deficient mice and Zdhhc13-knockdown Hep1-6 cells. These results indicate that ZDHHC13 is an important regulator of mitochondrial activity. Collectively, our study allows for a systematic view of S-palmitoylation for identification of ZDHHC13 substrates and demonstrates the role of ZDHHC13 in mitochondrial function and metabolism in liver.

Design and Synthesis of Ligand Efficient Dual Inhibitors of Janus Kinase (JAK) and Histone Deacetylase (HDAC) Based on Ruxolitinib and Vorinostat.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is <100 nM potent against JAK2 and HDAC11, and is selective for the JAK family against a panel of 97 kinases. Broad cellular antiproliferative potency of 24 is supported by demonstration of JAK-STAT and HDAC pathway blockade in hematological cell lines. Methyl analogue 45 has an even more selective profile. This study provides new leads for assessment of JAK and HDAC pathway dual inhibiton achieved with a single molecule.

Protein Palmitoylation by ZDHHC13 Protects Skin against Microbial-Driven Dermatitis.

Atopic dermatitis is a complex chronic inflammatory skin disorder that results from intimate interactions among genetic predisposition, host environment, skin barrier defects, and immunological factors. However, a clear genetic roadmap leading to atopic dermatitis remains to be fully explored. From a genome-wide mutagenesis screen, deficiency of ZDHHC13, a palmitoylacyl transferase, has previously been associated with skin and multitissue inflammatory phenotypes. Here, we report that ZDHHC13 is required for skin barrier integrity and that deficiency of ZDHHC13 renders mice susceptible to environmental bacteria, resulting in persistent skin inflammation and an atopic dermatitis-like disease. This phenotype is ameliorated in a germ-free environment and is also attenuated by antibiotic treatment, but not by deletion of the Rag1 gene, suggesting that a microbial factor triggers inflammation rather than intrinsic adaptive immunity. Furthermore, skin from ZDHHC13-deficient mice has both elevated levels of IL-33 and type 2 innate lymphoid cells, reinforcing the role of innate immunity in the development of atopic dermatitis. In summary, our study suggests that loss of ZDHHC13 in skin impairs the integrity of multiple barrier functions and leads to a dermatitis lesion in response to microbial encounters.

Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice.

Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.