The module triad: a novel network biology approach to utilize patients' multi-omics data for target discovery in ulcerative colitis.

Tumor necrosis factor-[Formula: see text] inhibitors (TNFi) have been a standard treatment in ulcerative colitis (UC) for nearly 20 years. However, insufficient response rate to TNFi therapies along with concerns around their immunogenicity and inconvenience of drug delivery through injections calls for development of UC drugs targeting alternative proteins. Here, we propose a multi-omic network biology method for prioritization of protein targets for UC treatment. Our method identifies network modules on the Human Interactome-a network of protein-protein interactions in human cells-consisting of genes contributing to the predisposition to UC (Genotype module), genes whose expression needs to be modulated to achieve low disease activity (Response module), and proteins whose perturbation alters expression of the Response module genes to a healthy state (Treatment module). Targets are prioritized based on their topological relevance to the Genotype module and functional similarity to the Treatment module. We demonstrate utility of our method in UC and other complex diseases by efficiently recovering the protein targets associated with compounds in clinical trials and on the market . The proposed method may help to reduce cost and time of drug development by offering a computational screening tool for identification of novel and repurposing therapeutic opportunities in UC and other complex diseases.

Isoform- and Phosphorylation-specific Multiplexed Quantitative Pharmacodynamics of Drugs Targeting PI3K and MAPK Signaling in Xenograft Models and Clinical Biopsies.

Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytic performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kß inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours postdose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection.

UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

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.

Dissociation of Golgi-associated DHHC-type Zinc Finger Protein (GODZ)- and Sertoli Cell Gene with a Zinc Finger Domain-ß (SERZ-ß)-mediated Palmitoylation by Loss of Function Analyses in Knock-out Mice.

The γ2 subunit of GABA type A receptors (GABAARs) is thought to be subject to palmitoylation by both Golgi-associated DHHC-type zinc finger protein (GODZ; also known as DHHC3) and its paralog Sertoli cell gene with a zinc finger domain-ß (SERZ-ß; DHHC7) based on overexpression of enzymes and substrates in heterologous cells. Here we have further investigated the substrate specificity of these enzymes by characterization of GODZ and SERZ-ß knock-out (KO) mice as well as double KO (DKO) neurons. Palmitoylation of γ2 and a second substrate, growth-associated protein of 43 kDa, that is independently implicated in trafficking of GABAARs was significantly reduced in brain of GODZ KO versus wild-type (WT) mice but unaltered in SERZ-ß KO mice. Accumulation of GABAARs at synapses, GABAergic innervation, and synaptic function were reduced in GODZ KO and DKO neurons to a similar extent, indicating that SERZ-ß does not contribute to palmitoylation or trafficking of GABAARs even in the absence of GODZ. Notably, these effects were seen only when mutant neurons were grown in competition with WT neurons, thereby mimicking conditions of shRNA-transfected neurons previously used to characterize GODZ. However, GABA-evoked whole-cell currents of DKO neurons and the GABAAR cell surface expression in DKO neurons and GODZ or SERZ-ß KO brain slices were unaltered, indicating that GODZ-mediated palmitoylation selectively controls the pool of receptors at synapses. The different substrate specificities of GODZ and SERZ-ß in vivo were correlated with their differential localization to cis- versus trans-Golgi compartment, a mechanism that was compromised by overexpression of GODZ.

Effect of glutamate side chain length on intrahelical glutamate-lysine ion pairing interactions.

Ion pairing interactions between oppositely charged amino acids are important for protein structure stability. Despite the apparent electrostatic nature of these interactions, the charged amino acids Lys, Arg, Glu, and Asp have a different number of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of Glu (and Asp) side chain length on ion pairing interactions, a series of 36 monomeric α-helical peptides containing Zbb-Xaa (i, i+3), (i, i+4), and (i, i+5) (Zbb = Aad, Glu, Asp; Xaa = Lys, Orn, Dab, Dap) sequence patterns were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. Peptides with Glu and Aad exhibited similar helicity and pH dependence, whereas peptides with Asp behaved distinctly different. The side chain interaction energetics were derived from the CD data using the nesting block method coupled with modified Lifson-Roig theory. At pH 7, no Zbb-Xaa (i, i+5) interaction was observed, regardless of side chain length (consistent with the helix geometry). Interestingly, only Lys was capable of supporting Zbb-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Zbb-Xaa (i, i+4) interactions. In particular, the magnitude of both Zbb(-)-Lys (i, i+4) and Zbb(-)-Orn (i, i+4) interaction energies followed the trend Asp > Glu > Aad. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb-Xaa (i, i+3) interactions involved the χ(1) dihedral combination (g+, g+) for the i and i+3 residues, whereas the Zbb-Xaa (i, i+4) interactions were supported by the χ(1) dihedral combination (t, g+) for the i and i+4 residues. These calculated low energy conformers were consistent with conformations of intrahelical Asp-Lys and Glu-Lys salt bridges in a nonredundant protein structure database. These results suggest that Asp and Glu provide natural variation, and lengthening the Glu side chain further to Aad does not furnish additional characteristics that Glu cannot supply.

Differentiation of CD8+ T cells into effector cells is enhanced by physiological range hyperthermia.

In this study, we asked whether exposure to different physiologically relevant temperatures (33°C, 37°C, and 39.5°C) could affect subsequent antigen-specific, activation-related events of naive CD8(+) T cells. We observed that temporary exposure of CD62L(hi)CD44(lo) Pmel-1 CD8(+) cells to 39.5°C prior to their antigen-dependent activation with gp100(25-33) peptide-pulsed C57BL/6 splenocytes resulted in a greater percentage of cells, which eventually differentiated into CD62L(lo)CD44(hi) effector cells compared with cells incubated at 33°C and 37°C. However, the proliferation rate of naive CD8(+) T cells was not affected by mild heating. While exploring these effects further, we observed that mild heating of CD8(+) T cells resulted in the reversible clustering of GM1(+) CD-microdomains in the plasma membrane. This could be attributable to a decrease in line tension in the plasma membrane, as we also observed an increase in membrane fluidity at higher temperatures. Importantly, this same clustering phenomenon was observed in CD8(+) T cells isolated from spleen, LNs, and peripheral blood following mild whole-body heating of mice. Further, we observed that mild heating also resulted in the clustering of TCRß and the CD8 coreceptor but not CD71R. Finally, we observed an enhanced rate of antigen-specific conjugate formation with APCs following mild heating, which could account for the difference in the extent of differentiation. Overall, these novel findings may help us to further understand the impact of physiologically relevant temperature shifts on the regulation of antigen-specific CD8(+) T cell activation and the subsequent generation of effector cells.

GABAA receptor trafficking-mediated plasticity of inhibitory synapses.

Proper developmental, neural cell-type-specific, and activity-dependent regulation of GABAergic transmission is essential for virtually all aspects of CNS function. The number of GABA(A) receptors in the postsynaptic membrane directly controls the efficacy of GABAergic synaptic transmission. Thus, regulated trafficking of GABA(A) receptors is essential for understanding brain function in both health and disease. Here we summarize recent progress in the understanding of mechanisms that allow dynamic adaptation of cell surface expression and postsynaptic accumulation and function of GABA(A) receptors. This includes activity-dependent and cell-type-specific changes in subunit gene expression, assembly of subunits into receptors, as well as exocytosis, endocytic recycling, diffusion dynamics, and degradation of GABA(A) receptors. In particular, we focus on the roles of receptor-interacting proteins, scaffold proteins, synaptic adhesion proteins, and enzymes that regulate the trafficking and function of receptors and associated proteins. In addition, we review neuropeptide signaling pathways that affect neural excitability through changes in GABA(A)R trafficking.