Geranylgeranyltransferase I mediates BDNF‐induced synaptogenesis

Geranylgeranyltransferase I (GGT) is a prenyltransferase that mediates lipid modification of Rho small GTPases, such as Rho, Rac, and Cdc42, which are important for neuronal synaptogenesis. Although GGT is expressed in brain extensively, the function of GGT in central nerves system is largely unknown so far. We have previously demonstrated that GGT promotes the basal and neuronal activity and brain‐derived neurotrophic factor (BDNF)‐induced dendritic morphogenesis of cultured hippocampal neurons and cerebellar slices. This study is to explore the function and mechanism of GGT in neuronal synaptogenesis. We found that the protein level and activity of GGT gradually increased in rat hippocampus from P7 to P28 and subcellular located at synapse of neurons. The linear density of Synapsin 1 and post‐synaptic density protein 95 increased by over‐expression of GGT β, while reduced by inhibition or down‐regulation of GGT. In addition, GGT and its known substrate Rac was activated by BDNF, which promotes synaptogenesis in cultured hippocampal neurons. Furthermore, BDNF‐induced synaptogenesis was eliminated by GGT inhibition or down‐regulation, as well as by non‐prenylated Rac1 over‐expression. Together, our data suggested that GGT mediates BDNF‐induced neuronal synaptogenesis through Rac1 activation.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.     

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K⁺ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.     

Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.     

Features of Pro-σK Important for Cleavage by SpoIVFB,an Intramembrane Metalloprotease

Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-σ^K during sporulation. To elucidate features of Pro-σ^K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-σ^K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-σ^K residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2′). These features appear to be conserved among Pro-σ^K orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2′ of Pro-σ^K. A Lys residue at P3′ of Pro-σ^K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9′). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σ^K. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.     

Ubiquitin E3 Ligase CRL4CDT2/DCAF2 as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4^CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4^CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.