Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction with IκBβ

Enrichment of human islets with Syntaxin 4 (STX4) improves functional β-cell mass through a nuclear factor- kB (NF-kB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. To determine the signaling events linking STX4 enrichment and downregulation of NF-kB activity, STX4 was overexpressed in human islets, EndoC-βH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1β, tumor necrosis factor-a and interferon-g, individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IkBβ but not IkBa. Inhibition of IKKβ prevented IkBβ degradation, suggesting that IKKβ phosphorylates IkBβ. Moreover, the IKKβ inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKβ in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IkBβ from proteasomal degradation, and like wildtype STX4, stabilized STX4 coimmunoprecipitated with IkBβ and the NF-kB p50 subunit. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-kB signaling in β-cells <i>via</i> associating with and preventing IkBβ degradation, suppressing chemokine expression, and protecting islet β-cells from cytokine-mediated dysfunction and demise.

Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction with IκBβ

Enrichment of human islets with Syntaxin 4 (STX4) improves functional β-cell mass through a nuclear factor- kB (NF-kB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. To determine the signaling events linking STX4 enrichment and downregulation of NF-kB activity, STX4 was overexpressed in human islets, EndoC-βH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1β, tumor necrosis factor-a and interferon-g, individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IkBβ but not IkBa. Inhibition of IKKβ prevented IkBβ degradation, suggesting that IKKβ phosphorylates IkBβ. Moreover, the IKKβ inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKβ in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IkBβ from proteasomal degradation, and like wildtype STX4, stabilized STX4 coimmunoprecipitated with IkBβ and the NF-kB p50 subunit. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-kB signaling in β-cells <i>via</i> associating with and preventing IkBβ degradation, suppressing chemokine expression, and protecting islet β-cells from cytokine-mediated dysfunction and demise.

Transcriptomic and Proteostasis Networks of CFTR and the Development of Small Molecule Modulators for the Treatment of Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The diversity of mutations and the multiple ways by which the protein is affected present challenges for therapeutic development. The observation that the Phe508del-CFTR mutant protein is temperature sensitive provided proof of principle that mutant CFTR could escape proteosomal degradation and retain partial function. Several specific protein interactors and quality control checkpoints encountered by CFTR during its proteostasis have been investigated for therapeutic purposes, but remain incompletely understood. Furthermore, pharmacological manipulation of many CFTR interactors has not been thoroughly investigated for the rescue of Phe508del-CFTR. However, high-throughput screening technologies helped identify several small molecule modulators that rescue CFTR from proteosomal degradation and restore partial function to the protein. Here, we discuss the current state of CFTR transcriptomic and biogenesis research and small molecule therapy development. We also review recent progress in CFTR proteostasis modulators and discuss how such treatments could complement current FDA-approved small molecules.

Phosphorylation of Tyr sites of USP1 protein in K562 cell as a progression factor of chronic myeloid leukemia

Aim. To study the properties of Tyr phosphorylation of USP1 protein in K562 cells. Methods. The bioinformatics analysis of the USP1 protein sites of phosphorylayion using the Phosphosite software. Coimmunoprecipitation, Western blot. Immunofluorescence analysis and confocal microscopy. Results. Potential phosphorylation sites for USP1 protein for Tyr are provided. Phosphorylated form of USP1 protein detected in K562 cells. Using immunofluorescence analysis and confocal microscopy, we found that Tyr phosphorylated forms of USP1 protein are localized in the nucleus. Conclusions. We deem that Tyr phosphorylation of USP1 protein is the consequence of its interaction with Bcr-Abl oncoprotein, which has high kinase activity. USP1 phosphorylation can raise deubiquitinating activity of this protein, and as a result, avert the proteosomal degradation of Bcr-Abl in cell and facilitate the progress of the disease.Keywords: chronic myeloid leukemia, Bcr-Abl, USP1, Tyr site of phosphorylation.

Different SUMO Paralogs Determine the Fate of WT and Mutant CFTRs: Biogenesis vs. Degradation

ABSTRACTA pathway for CFTR degradation is initiated by Hsp27 which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogs form poly-chains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, PIAS4, which increased WT and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR three-fold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation 3-fold and stabilized mature WT CFTR at the PM. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1in vivoand reduced its conjugation to SUMO-2/3. These SUMO paralog specific effects of PIAS4 were reproducedin vitrousing purified F508del NBD1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ~50%, and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogs determine the fates of WT and mutant CFTRs, and they suggest that a paralog switch during biogenesis can direct these proteins to different outcomes: biogenesis vs. degradation.

Inhibition of Hsp70 suppresses neuronal hyperexcitability and attenuates seizures by enhancing A-type potassium currents

ABSTRACTThe heat shock protein 70 (Hsp70) is upregulated in response to stress and has been implicated as a stress marker in temporal lobe epilepsy (TLE). However, whether Hsp70 plays a pathologic or protective role in TLE remains unclear. Here we report that Hsp70 exerts an unexpected deleterious role in kainic acid (KA)-induced seizures, and inhibition of Hsp70 suppresses neuronal hyperexcitability and attenuates both acute and chronic seizures via enhancing A-type potassium currents primarily formed by Kv4 α-subunits and auxiliary KChIPs. Proteosomal degradation of Kv4-KChIP4a channel complexes is enhanced by Hsp70, which can be reversed by the Hsp70 inhibitors, 2-phenylethynesulfonamide (PES) and VER-155008 (VER). In cultured hippocampal neurons, either PES or VER can increase A-type Kv4 current to suppress neuronal hyperexcitability. Mechanistically, Hsp70-CHIP complexes directly bind to the N-terminus of auxiliary KChIP4a and target Kv4-KChIP4a complexes to the proteasome. Our findings reveal a previously unrecognized role of Hsp70 in mediating degradation of Kv4-KChIP4a complexes and regulating neuronal excitability, thus highlighting a therapeutic potential for hyperexcitability-related neurological disorders through Hsp70 inhibition.