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C-terminal splice variants of P/Q-type Ca2+ channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins

Hirano M, Takada Y, Wong CF, Yamaguchi K, Kotani H, Kurokawa T, et al.

J Biol Chem, 2017 06 02;292(22):9365-9381.
PMID: 28377503 DOI: 10.1074/jbc.M117.778829

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.

Matched MeSH terms: GTP-Binding Proteins/metabolism*
Proteomic analysis reveals brain Rab35 as a potential biomarker of mitragynine withdrawal in rats

Hassan R, Othman N, Mansor SM, Müller CP, Hassan Z

Brain Res Bull, 2021 07;172:139-150.
PMID: 33901587 DOI: 10.1016/j.brainresbull.2021.04.018

Mitragyna speciosa, also known as kratom, has been used for mitigating the severity of opioid withdrawal in humans. Its main indole alkaloid, mitragynine, has been considered as a pharmacotherapy for pain conditions and opioid replacement therapy. However, at high doses, chronic mitragynine may also have an addiction potential. The effects of chronic action of mitragynine in the brain are still unknown. The present study developed a mitragynine withdrawal model in rats and used it for a proteomic analysis of mitragynine withdrawal effects. Mitragynine (30 mg/kg, i.p.) was administered daily over a period of 14 days and then withdrawn. A proteomic analysis revealed that from a total of 1524 proteins identified, 31 proteins were upregulated, and 3 proteins were downregulated in the mitragynine withdrawal model. The Rab35 protein expression increased most profoundly in the mitragynine withdrawal group as compared to vehicle group. Therefore, it is proposed that Rab35 in the brain might be considered as a potential biomarker during mitragynine withdrawal and might be valuable target protein in developing new pharmacotherapies in the future.

Matched MeSH terms: rab GTP-Binding Proteins/metabolism*
Human neuronal cell protein responses to Nipah virus infection

Chang LY, Ali AR, Hassan SS, AbuBakar S

Virol J, 2007;4:54.
PMID: 17553172

Nipah virus (NiV), a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches.

Matched MeSH terms: GTP-Binding Proteins/metabolism
Restitution of epithelial cells during intestinal mucosal wound healing: The effect of a polysaccharide from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden

Veeraperumal S, Qiu HM, Tan CS, Ng ST, Zhang W, Tang S, et al.

J Ethnopharmacol, 2021 Jun 28;274:114024.
PMID: 33727110 DOI: 10.1016/j.jep.2021.114024

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerotis (Cooke) Ryvarden cultivar TM02, also known as tiger's milk mushroom, is regarded as important folk medicine in Malaysia, while is used for the treatment of liver cancer, chronic hepatitis, gastric ulcer in traditional Chinese medicine. However, there is no compilation of scientific evidence that its protection for gastric, and no attempts have been made to understand how polysaccharides in Lignosus rhinocerotis might promote intestinal mucosal wound healing.
AIM OF THE STUDY: This study aimed to investigate the effect and mechanism of β-glucan prepared from L. rhinocerotis using an enzymatic method on epithelial restitution during intestinal mucosal damage.
MATERIALS AND METHODS: Based on FT-IR, MALDI-TOF-MS, HPSEC-MALLS-RID, and AFM, the structure of polysaccharides from L. rhinocerotis was analysed. In addition, polysaccharides were used to test for wound healing activity in IEC-6 cells by measuring cell migration, proliferation, and expression of cell division control protein 42, Rac-1, RhoA, and Par-3.
RESULTS: β-glucan was extracted using enzyme-assisted extraction, and a yield of approximately 8.5 ± 0.8% was obtained from the dried biomass. The β-glucan extracted by enzyme-assisted extraction (EAE) of polysaccharides was composed entirely of D-glucose with a total carbohydrate content of 95.5 ± 3.2%. The results of HPLC, FTIR, and MALDI-TOF-MS analyses revealed EAEP to be confirmed as β-glucan. The molecular weight of prepared β-glucan was found to be 5.315 × 104 g/mol by HPSEC-MALLS-RID. Furthermore, mucosal wound healing studies showed that the treatment of IEC-6 with a β-glucan concentration of 200 μg/mL promoted cell migration and proliferation, and it enhanced the protein expression of cell division control protein 42, Rac-1, RhoA, and Par-3.
CONCLUSIONS: The present study reveals that the prepared β-glucan accelerates intestinal epithelial cell proliferation and migration via activation of Rho-dependent pathway. Hence, β-glucan can be employed as a prospective therapeutic agent for the treatment of diseases associated with gastrointestinal mucosal damage, such as peptic ulcers and inflammatory bowel disease.

Matched MeSH terms: rho GTP-Binding Proteins/metabolism
Fulltext Edible bird's nest modulate intracellular molecular pathways of influenza A virus infected cells

Haghani A, Mehrbod P, Safi N, Kadir FA, Omar AR, Ideris A

BMC Complement Altern Med, 2017 Jan 05;17(1):22.
PMID: 28056926 DOI: 10.1186/s12906-016-1498-x

Edible Bird's Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized.

Matched MeSH terms: rab5 GTP-Binding Proteins/metabolism
Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma

Zanaruddin SN, Saleh A, Yang YH, Hamid S, Mustafa WM, Khairul Bariah AA, et al.

Hum Pathol, 2013 Mar;44(3):417-26.
PMID: 23026198 DOI: 10.1016/j.humpath.2012.06.007

The presence of lymph node (LN) metastasis significantly affects the survival of patients with oral squamous cell carcinoma (OSCC). Successful detection and removal of positive LNs are crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the LNs, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis-positive LNs in the neck. We developed a molecular signature in the primary tumor that could predict LN metastasis in OSCC. A total of 211 cores from 55 individuals were included in the study. Eleven proteins were evaluated using immunohistochemical analysis in a tissue microarray. Of the 11 biomarkers evaluated using receiver operating curve analysis, epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2/neu), laminin, gamma 2 (LAMC2), and ras homolog family member C (RHOC) were found to be significantly associated with the presence of LN metastasis. Unsupervised hierarchical clustering-demonstrated expression patterns of these 4 proteins could be used to differentiate specimens that have positive LN metastasis from those that are negative for LN metastasis. Collectively, EGFR, HER-2/neu, LAMC2, and RHOC have a specificity of 87.5% and a sensitivity of 70%, with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated that the LN signature could independently predict disease-specific survival (P = .036). The 4-protein LN signature validated in an independent set of samples strongly suggests that it could reliably distinguish patients with LN metastasis from those who were metastasis-free and therefore could be a prognostic tool for the management of patients with OSCC.

Matched MeSH terms: rho GTP-Binding Proteins/metabolism
Fulltext Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy

Miyake N, Fukai R, Ohba C, Chihara T, Miura M, Shimizu H, et al.

Am J Hum Genet, 2016 Oct 06;99(4):950-961.
PMID: 27666374 DOI: 10.1016/j.ajhg.2016.08.005

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.

Matched MeSH terms: GTP-Binding Proteins/metabolism
Fulltext Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins

Ng YL, Olivos-García A, Lim TK, Noordin R, Lin Q, Othman N

Am J Trop Med Hyg, 2018 12;99(6):1518-1529.
PMID: 30298805 DOI: 10.4269/ajtmh.18-0415

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

Matched MeSH terms: rho GTP-Binding Proteins/metabolism
Regulatory actions of 3',5'-cyclic adenosine monophosphate on osteoclast function: possible roles of Epac-mediated signaling

Jeevaratnam K, Salvage SC, Li M, Huang CL

Ann N Y Acad Sci, 2018 Dec;1433(1):18-28.
PMID: 29846007 DOI: 10.1111/nyas.13861

Alterations in cellular levels of the second messenger 3',5'-cyclic adenosine monophosphate ([cAMP]i ) regulate a wide range of physiologically important cellular signaling processes in numerous cell types. Osteoclasts are terminally differentiated, multinucleated cells specialized for bone resorption. Their systemic regulator, calcitonin, triggers morphometrically and pharmacologically distinct retraction (R) and quiescence (Q) effects on cell-spread area and protrusion-retraction motility, respectively, paralleling its inhibition of bone resorption. Q effects were reproduced by cholera toxin-mediated Gs -protein activation known to increase [cAMP]i , unaccompanied by the [Ca2+ ]i changes contrastingly associated with R effects. We explore a hypothesis implicating cAMP signaling involving guanine nucleotide-exchange activation of the small GTPase Ras-proximate-1 (Rap1) by exchange proteins directly activated by cAMP (Epac). Rap1 activates integrin clustering, cell adhesion to bone matrix, associated cytoskeletal modifications and signaling processes, and transmembrane transduction functions. Epac activation enhanced, whereas Epac inhibition or shRNA-mediated knockdown compromised, the appearance of markers for osteoclast differentiation and motility following stimulation by receptor activator of nuclear factor kappa-Β ligand (RANKL). Deficiencies in talin and Rap1 compromised in vivo bone resorption, producing osteopetrotic phenotypes in genetically modified murine models. Translational implications of an Epac-Rap1 signaling hypothesis in relationship to N-bisphosphonate actions on prenylation and membrane localization of small GTPases are discussed.

Matched MeSH terms: rap1 GTP-Binding Proteins/metabolism
Fulltext Simvastatin modulates cellular components in influenza A virus-infected cells

Mehrbod P, Hair-Bejo M, Tengku Ibrahim TA, Omar AR, El Zowalaty M, Ajdari Z, et al.

Int J Mol Med, 2014 Jul;34(1):61-73.
PMID: 24788303 DOI: 10.3892/ijmm.2014.1761

Influenza A virus is one of the most important health risks that lead to significant respiratory infections. Continuous antigenic changes and lack of promising vaccines are the reasons for the unsuccessful treatment of influenza. Statins are pleiotropic drugs that have recently served as anti-influenza agents due to their anti-inflammatory activity. In this study, the effect of simvastatin on influenza A-infected cells was investigated. Based on the MTT cytotoxicity test, hemagglutination (HA) assay and qPCR it was found that simvastatin maintained cell viability and decreased the viral load significantly as compared to virus-inoculated cells. The expression of important pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interferon-γ), which was quantified using ELISA showed that simvastatin decreased the expression of pro-inflammatory cytokines to an average of 2-fold. Furthermore, the modulation of actin filament polymerization was determined using rhodamine staining. Endocytosis and autophagy processes were examined by detecting Rab and RhoA GTPase protein prenylation and LC3 lipidation using western blotting. The results showed that inhibiting GTPase and LC3 membrane localization using simvastatin inhibits influenza replication. Findings of this study provide evidence that modulation of RhoA, Rabs and LC3 may be the underlying mechanisms for the inhibitory effects of simvastatin as an anti-influenza compound.

Matched MeSH terms: rab GTP-Binding Proteins/metabolism

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