DHHC17 Is a New Regulator of AMPK Signaling.

The association of AMP-activated protein kinase (AMPK) with membranes plays a critical role in the regulation of AMPK activation and function. Protein lipid modification, including palmitoylation, myristoylation, and farnesyation, constitutes a crucial mechanism in the regulation of protein dynamic interactions with membranes. Among the three subunits of the AMPK heterotrimeric complex, the structural subunit AMPKß is myristoylated and the catalytic subunit AMPKα is palmitoylated. Here, we report the characterization of AMPKα palmitoylation. We found that AMKPα was palmitoylated at Cys209 and Cys543, and this was required for AMPK activation and subcellular membrane compartmentalization. To understand the regulation of AMPKα palmitoylation, we have identified DHHC17 as a candidate palmitoyltransferase for AMPKα and found that DHHC17, by palmitoylating AMPKα, modulated AMPK membrane association and activation in response to energy stress. To determine the role of DHHC17 in cell function, we generated DHHC17 liver-specific knockout mice and found that inactivation of DHHC17 in the mouse liver impaired AMPK activation and hepatic autophagy and caused a type 2 diabetes-like syndrome. Overall, our studies demonstrate that AMPKα palmitoylation plays a critical role in AMPK activation and that DHHC17, through its modulation of AMPK signaling, constitutes a new regulator of hepatic metabolism.

In Situ Saturating Mutagenesis Screening Identifies a Functional Genomic Locus that Regulates Ucp1 Expression.

A better understanding of the molecular mechanisms that control the UCP1 expression in brown and beige adipocytes is essential for us to modulate adipose cell fate and promote thermogenesis, which may provide a therapeutic view for the treatment of obesity and obesity-related diseases. To systematically identify cis-element(s) that transcriptionally regulates Ucp1, we here took advantage of the high-throughput CRIPSR-Cas9 screening system, and performed an in situ saturating mutagenesis screen, by using a customized sgRNA library targeting the ~ 20 kb genomic region near Ucp1. Through the screening, we have identified several genomic loci that may contain key regulatory element for Ucp1 expression in cultured brown and white adipocytes in vitro, and in inguinal white adipose tissue in vivo. Our study highlights a broadly useful approach for studying cis-regulatory elements in a high-throughput manner.

Gain-of-Function Mutations of SLC16A11 Contribute to the Pathogenesis of Type 2 Diabetes.

DNA variants in the SLC16A11 coding region were identified to be strongly associated with type 2 diabetes (T2DM) in a Mexican population. Previous studies suggested that these variants disrupt SLC16A11 function and therefore proposed to revive SLC16A11 levels or activity to achieve therapeutic benefit. However, with knockout mouse models, here we show that Slc16a11 depletion has no significant metabolic defects. Further studies demonstrate that reconstitution of the mutant, but not the wild-type Slc16a11, in the liver of knockout mice causes more triglyceride accumulation and induction of insulin resistance via upregulation of lipin 1, suggesting gaining of aberrant functions of the mutant protein that affects lipid metabolism. Our findings offer a different explanation to the function of these diabetic variants, challenging the concept of enhancing SLC16A11 function to treat T2DM. The contradictory results by our and previous studies suggest that how the SLC16A11 locus contributes to human metabolism warrants further investigation.

Screening of FDA-approved drugs identifies sutent as a modulator of UCP1 expression in brown adipose tissue.

BACKGROUND: The pharmacological activation of thermogenesis in brown adipose tissue has long been considered promising strategies to treat obesity. However, identification of safe and effective agents remains a challenge. In this study, we addressed this challenge by developing a cellular system with a fluorescence readout, and applied in a high-throughput manner to screen for FDA-approved drugs that may activate endogenous UCP1 expression in adipocytes. METHODS: We have generated a Ucp1-2A-GFP reporter mouse, in which GFP intensity serves as a surrogate of the endogenous expression level of UCP1 protein; and immortalized brown adipocytes were derived from this mouse model and applied in drug screening. Candidate drugs were further tested in mouse models either fed with normal chow or high fat diet to induce obesity. FINDINGS: By using the cellular screening platform, we identified a group of FDA-approved drugs that can upregulate UCP1 expression in brown adipocyte, including previously known UCP1 activators and new candidate drugs. Further studies focusing on a previously unreported drug-sutent, revealed that sutent treatment could increase the energy expenditure and inhibit lipid synthesis in mouse adipose and liver tissues, resulting in improved metabolism and resistance to obesity. INTERPRETATION: This study offered an easy-to-use cellular screening system for UCP1 activators, and provided a candidate list of FDA-approved drugs that can potentially treat obesity. Further study of these candidates may shed new light on the drug discovery towards obesity. FUND: National Key Research and Development Program and the Strategic Priority Research Program of the Chinese Academy of Sciences, etc. (250 words).

Genome engineering of stem cell organoids for disease modeling.

Precision medicine emerges as a new approach that takes into account individual variability. Successful realization of precision medicine requires disease models that are able to incorporate personalized disease information and recapitulate disease development processes at the molecular, cellular and organ levels. With recent development in stem cell field, a variety of tissue organoids can be derived from patient specific pluripotent stem cells and adult stem cells. In combination with the state-of-the-art genome editing tools, organoids can be further engineered to mimic disease-relevant genetic and epigenetic status of a patient. This has therefore enabled a rapid expansion of sophisticated in vitro disease models, offering a unique system for fundamental and biomedical research as well as the development of personalized medicine. Here we summarize some of the latest advances and future perspectives in engineering stem cell organoids for human disease modeling.

DHHC7 Palmitoylates Glucose Transporter 4 (Glut4) and Regulates Glut4 Membrane Translocation.

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane plays a key role in the dynamic regulation of glucose homeostasis. We recently showed that this process is critically dependent on palmitoylation of Glut4 at Cys-223. To gain further insights into the regulation of Glut4 palmitoylation, we set out to identify the palmitoyl acyltransferase (PAT) involved. Here we report that among 23 mammalian DHHC proteins, DHHC7 is the major Glut4 PAT, based on evidence that ectopic expression of DHHC7 increased Glut4 palmitoylation, whereas DHHC7 knockdown in 3T3-L1 adipocytes and DHHC7 KO in adipose tissue and muscle decreased Glut4 palmitoylation. Moreover, inactivation of DHHC7 suppressed insulin-dependent Glut4 membrane translocation in both 3T3-L1 adipocytes and primary adipocytes. Finally, DHHC7 KO mice developed hyperglycemia and glucose intolerance, thereby confirming that DHHC7 represents the principal PAT for Glut4 and that this mechanism is essential for insulin-regulated glucose homeostasis.

Elmo2 Is a Regulator of Insulin-dependent Glut4 Membrane Translocation.

Elmo2, a member of the Elmo protein family, has been implicated in the regulation of Rac1 and Akt activation. Recently, we found that Elmo2 specifically interacts with ClipR-59. Because Akt and Rac1 have been implicated in insulin dependent Glut4 membrane translocation, we hypothesize here that Elmo2 may play a role in insulin-dependent Glut4 membrane translocation. Accordingly, we found that overexpression of Elmo2 enhanced, whereas its knockdown suppressed, insulin-dependent Glut4 membrane translocation in both 3T3-L1 adipocytes and L6 skeletal muscle cells. We also examined whether Elmo2 contributes to the insulin-mediated activation of Rac1 and Akt. We found that Elmo2 is required for insulin-induced Rac1 GTP loading, but not AKT activation, in L6 cells induced by insulin. Instead, Elmo2 is required to promote the insulin-induced membrane association of Akt. Together, our studies demonstrate that Elmo2 is a new regulator of insulin-dependent Glut4 membrane translocation through modulating Rac1 activity and Akt membrane compartmentalization.

Glut4 palmitoylation at Cys223 plays a critical role in Glut4 membrane trafficking.

Recently, we identified Glut4 as a palmitoylated protein in adipocytes. To understand the role of Glut4 palmitoylation in Glut4 membrane trafficking, a process that is essential for maintenance of whole body glucose homeostasis, we have characterized Glut4 palmitoylation. We found that Glut4 is palmitoylated at Cys223 and Glut4 palmitoylation at Cys223 is essential for insulin dependent Glut4 membrane translocation as substitution of Cys223 with a serine residue in Glut4 (C223S Glut4) diminished Glut4 responsiveness to insulin in membrane translocation in both adipocytes and CHO-IR cells. We have examined C223S Glut4 subcellular localization and observed that it was absence from tubular-vesicle structure, where insulin responsive Glut4 vesicles were presented. Together, our studies uncover a novel mechanism under which Glut4 palmitoylation regulates Glut4 sorting to insulin responsive vesicles, thereby insulin-dependent Glut4 membrane translocation.

Expression of recombinant human lysozyme in egg whites of transgenic hens.

Chicken egg lysozyme (cLY) is an enzyme with 129 amino acid (AA) residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV) infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY) cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY) at 57.66 ± 4.10 µg/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 µg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.

ClipR-59 interacts with Elmo2 and modulates myoblast fusion.

Recent studies using ClipR-59 knock-out mice implicated this protein in the regulation of muscle function. In this report, we have examined the role of ClipR-59 in muscle differentiation and found that ClipR-59 knockdown in C2C12 cells suppressed myoblast fusion. To elucidate the molecular mechanism whereby ClipR-59 regulates myoblast fusion, we carried out a yeast two-hybrid screen using ClipR-59 as the bait and identified Elmo2, a member of the Engulfment and cell motility protein family, as a novel ClipR-59-associated protein. We showed that the interaction between ClipR-59 and Elmo2 was mediated by the atypical PH domain of Elmo2 and the Glu-Pro-rich domain of ClipR-59 and regulated by Rho-GTPase. We have examined the impact of ClipR-59 on Elmo2 downstream signaling and found that interaction of ClipR-59 with Elmo2 enhanced Rac1 activation. Collectively, our studies demonstrate that formation of an Elmo2·ClipR-59 complex plays an important role in myoblast fusion.

DHHC17 palmitoylates ClipR-59 and modulates ClipR-59 association with the plasma membrane.

ClipR-59 interacts with Akt and regulates Akt compartmentalization and Glut4 membrane trafficking in a plasma membrane association-dependent manner. The association of ClipR-59 with plasma membrane is mediated by ClipR-59 palmitoylation at Cys534 and Cys535. To understand the regulation of ClipR-59 palmitoylation, we have examined all known mammalian DHHC palmitoyltransferases with respect to their ability to promote ClipR-59 palmitoylation. We found that, among 23 mammalian DHHC palmitoyltransferases, DHHC17 is the major ClipR-59 palmitoyltransferase, as evidenced by the fact that DHHC17 interacted with ClipR-59 and palmitoylated ClipR-59 at Cys534 and Cys535. By palmitoylating ClipR-59, DHHC17 directly regulates ClipR-59 plasma membrane association, as ectopic expression of DHHC17 increased whereas silencing of DHHC17 reduced the levels of ClipR-59 associated with plasma membrane. We have also examined the role of DHHC17 in Akt signaling and found that silencing of DHHC17 in 3T3-L1 adipocytes decreased the levels of Akt as well as ClipR-59 on the plasma membrane and impaired insulin-dependent Glut4 membrane translocation. We suggest that DHHC17 is a ClipR-59 palmitoyltransferase that modulates ClipR-59 plasma membrane binding, thereby regulating Akt signaling and Glut4 membrane translocation in adipocytes.

Interaction of constitutive photomorphogenesis 1 protein with protein-tyrosine phosphatase 1B suppresses protein-tyrosine phosphatase 1B activity and enhances insulin signaling.

Recent studies reveal that COP1 suppresses the expression of gluconeogenetic genes and prohibits hepatic glucose production. To get more insight into COP1 in hepatic cells, we examined the impact of COP1 on insulin-responsive genes and insulin signaling. We found that COP1 increased the responsiveness of insulin-modulated genes to insulin in that it promoted the expression of insulin-induced genes and inhibited that of insulin-suppressed genes and that COP1 enhanced insulin signaling as it promoted phosphorylation of Akt and ERK as well as tyrosine phosphorylation of IRß induced by insulin. To delineate the mechanism under which COP1 modulates insulin signaling, we examined the possibility that COP1 modulates the activity of PTP1B, the major insulin receptor tyrosine phosphatase. The results indicated that COP1 physically interacted with PTP1B and suppressed PTP1B phosphatase activity as well as the association of PTP1B with IRß. We suggest that COP1 is a positive regulator of hepatic insulin signaling.

Assembly and in vitro functional analysis of zinc finger nuclease specific to the 3' untranslated region of chicken ovalbumin gene.

Synthetic zinc finger nucleases (ZFNs) are useful for the improvement of site directed integration of foreign gene into vertebrate chromosomes. To facilitate site-directed integration of foreign genes into the 3'-untranslated region of the chicken ovalbumin gene, we have constructed ZFN expression vectors using Zinc Finger Consortium Vector Kits and tested the functionality of these ZFN constructs. Coding sequences for 6 zinc fingers were assembled following the modular assembly method. The zinc finger assembly was fused to two FokI catalytic domains. Various configurations of linker regions between domains were tested for their influence on enzymatic activity, using plasmid substrate containing the target sequence. Results indicated that ZFN with an elongated linker between two nuclease domains had a high catalytic activity.