Investigating the Functional Interaction between RhoGDI Family Proteins and Activated Cdc42 associated-kinase (ACK)

ACK is a non-receptor tyrosine kinase and an effector protein for Cdc42. ACK has been implicated in carcinogenesis events especially in promoting cell migration and invasion, indicating a need for a fuller understanding of the cellular roles of ACK both in normal and pathological conditions. A previous yeast-2-hybrid screen in the lab identified RhoGDI-3 as an interacting partner for ACK. RhoGDI-3 is a member of RhoGDI family of proteins, which are negative regulators that maintains Rho-family GTPases in the inactive GDP-bound state and sequesters them in the cytosol. RhoGDIs have also been found to become deregulated in cancer and it is possible therefore that they play role in ACK-driven cancer progression. In this work, it is shown that ACK binds but does not phosphorylate the RhoGDIs in cells or in vitro. ACK was shown to shuttle between the cytoplasm and the nucleus. RhoGDI-1 and -2 have been found only in the cytoplasm, while RhoGDI-3 is localized to both cytoplasm and the nucleus, under the conditions tested. The interaction between RhoGDI-3 and ACK occurs predominantly in the nucleus and RhoGDI-3 levels were shown to decrease following co-expression with ACK, especially in the nucleus. Data generated here shows that ACK-mediates RhoGDI-3 proteasomal degradation potentially by regulating RhoGDI-3 ubiquitination. In order to determine the cellular effects of the interaction between ACK and the RhoGDIs, all possible Rho-family GTPases that interact with the RhoGDIs were determined in a systematic study. RhoGDI-1 and 2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity interacting with several novel interacting partners within the Rho, Rac and Cdc42 subfamilies. Unexpectedly RhoGDI-3 was found to form complexes with the atypical Rho GTPases such as RhoD, Wrch2, Rnd2, Miro2 and RhoH which are not regulated by standard GDP/GTP cycling. The GTP levels of these target proteins were found to decrease following co-expression with RhoGDI-3, confirming its role as a negative regulator of these Rho GTPases. ACK was shown to regulate the activation of these target proteins, including RhoA, RhoB, Rac1 and RhoH, which are known to be involved in cell proliferation and migration. Aberrant activation of these target protein is frequently observed in cancer, suggesting a role for RhoGDI to drive ACK oncogenicity.Ministry of Higher Education Malaysia and Universiti Sains Malaysi

Antiulcer properties of Kelulut honey against ethanol-induced gastric ulcer

Ulcers in the gastrointestinal tract refer to any appreciable depth of break in the mucosa lining that may involve submucosa. Common types of ulcer include peptic, gastric and duodenal ulcer, which may lead to chronic inflammation. Ulcers may be caused by excessive alcohol intake or prolonged use of non-steroidal anti-inflammatory drugs (NSAID), in addition to several other factors. Conventional medication such as Omeprazole (proton pump inhibitor) and Ranitidine (H2 blockers) for management of ulcers may cause severe side effects such as myelosupression and abnormal heart rhythm. This has driven researchers to explore the potential of natural products for management of ulcers with reduced side effects. Kelulut honey (KH) is a type of honey that is produced by stingless bees from the Trigona species. It is believed to have a lot of medicinal properties such as being antimicrobial, antioxidant and antidiabetic. Yet, no scientific study has been carried out on its antiulcer properties. This study was carried out to determine the antiulcer properties of KH. Eighteen male Sprague dawley rats (5 to 6 weeks old, weighing between 200 and 300 g) were divided into three groups (n=6). The groups were 1) normal control group (without ulcer, without KH), 2) positive control group (with ulcer, without KH) and 3) treatment group (with ulcer, treated with KH). The treatment, KH (1183 mg/kg), was given twice daily for 30 consecutive days by oral administration. On Day 31, the rats were induced with absolute ethanol (5 mL/kg) via oral administration after being fasted for 24 h and were sacrificed 15 min after the induction. The stomach was collected for macroscopic and histopathological evaluation. Pretreatment with KH significantly reduced (p<0.05) both the total area of ulcer and the ulcer index compared to the positive control group. The percentage of ulcer inhibition in the KH pre-treated group was 65.56% compared with the positive control group. The treatment, KH, exhibited antiulcer properties against ethanol-induced gastric ulcer

The Role of RhoH in TCR Signalling and Its Involvement in Diseases

As an atypical member of the Rho family small GTPases, RhoH shares less than 50% sequence similarity with other members, and its expression is commonly observed in the haematopoietic lineage. To date, RhoH function was observed in regulating T cell receptor signalling, and less is known in other haematopoietic cells. Its activation may not rely on the standard GDP/GTP cycling of small G proteins and is thought to be constitutively active because critical amino acids involved in GTP hydrolysis are absent. Alternatively, its activation can be regulated by other types of regulation, including lysosomal degradation, somatic mutation and transcriptional repressor, which also results in an altered protein expression. Aberrant protein expression of RhoH has been implicated not only in B cell malignancies but also in immune-related diseases, such as primary immunodeficiencies, systemic lupus erythematosus and psoriasis, wherein its involvement may provide the link between immune-related diseases and cancer. RhoH association with these diseases involves several other players, including its interacting partner, ZAP−70; activation regulators, Vav1 and RhoGDI and other small GTPases, such as RhoA, Rac1 and Cdc42. As such, RhoH and its associated proteins are potential attack points, especially in the treatment of cancer and immune-related diseases

Molecular subversion of Cdc42 signalling in cancer.

Cdc42 is a member of the Rho family of small GTPases and a master regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. This small G protein and its regulators have been the subject of many years of fruitful investigation and the advent of functional genomics and proteomics has opened up new avenues of exploration including how it functions at specific locations in the cell. This has coincided with the introduction of new structural techniques with the ability to study small GTPases in the context of the membrane. The role of Cdc42 in cancer is well established but the molecular details of its action are still being uncovered. Here we review alterations found to Cdc42 itself and to key components of the signal transduction pathways it controls in cancer. Given the challenges encountered with targeting small G proteins directly therapeutically, it is arguably the regulators of Cdc42 and the effector signalling pathways downstream of the small G protein which will be the most tractable targets for therapeutic intervention. These will require interrogation in order to fully understand the global signalling contribution of Cdc42, unlock the potential for mapping new signalling axes and ultimately produce inhibitors of Cdc42 driven signalling.BBSR

The role of RhoH in TCR signalling and its involvement in diseases

As an atypical member of the Rho family small GTPases, RhoH shares less than 50% sequence similarity with other members, and its expression is commonly observed in the haematopoietic lineage. To date, RhoH function was observed in regulating T cell receptor signalling, and less is known in other haematopoietic cells. Its activation may not rely on the standard GDP/GTP cycling of small G proteins and is thought to be constitutively active because critical amino acids involved in GTP hydrolysis are absent. Alternatively, its activation can be regulated by other types of regulation, including lysosomal degradation, somatic mutation and transcriptional repressor, which also results in an altered protein expression. Aberrant protein expression of RhoH has been implicated not only in B cell malignancies but also in immune-related diseases, such as primary immunodeficiencies, systemic lupus erythematosus and psoriasis, wherein its involvement may provide the link between immune-related diseases and cancer. RhoH association with these diseases involves several other players, including its interacting partner, ZAP−70; activation regulators, Vav1 and RhoGDI and other small GTPases, such as RhoA, Rac1 and Cdc42. As such, RhoH and its associated proteins are potential attack points, especially in the treatment of cancer and immune-related diseases

A Complete Survey of RhoGDI Targets Reveals Novel Interactions with Atypical Small GTPases.

There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.This research was supported by a studentship to AMbAM from the Ministry of Higher Education, Malaysia, a BBSRC Doctoral Training Partnership studentship (BB/JO1454/1) to MH, an Isaac Newton Trust Research Grant Award (13.07(b)) to DO and HRM and an MRC project grant (G0700057) to DO and HR

Carbapenem Resistance among Marine Bacteria—An Emerging Threat to the Global Health Sector

The emergence of antibiotic resistance among pathogenic microorganisms is a major issue for global public health, as it results in acute or chronic infections, debilitating diseases, and mortality. Of particular concern is the rapid and common spread of carbapenem resistance in healthcare settings. Carbapenems are a class of critical antibiotics reserved for treatment against multidrug-resistant microorganisms, and resistance to this antibiotic may result in limited treatment against infections. In addition to in clinical facilities, carbapenem resistance has also been identified in aquatic niches, including marine environments. Various carbapenem-resistant genes (CRGs) have been detected in different marine settings, with the majority of the genes incorporated in mobile genetic elements, i.e., transposons or plasmids, which may contribute to efficient genetic transfer. This review highlights the potential of the marine environment as a reservoir for carbapenem resistance and provides a general overview of CRG transmission among marine microbes

Optimization of ionic liquid-based aqueous two-phase system for partial purification of bacteriocin-like inhibitory substance from Enterococcus faecium CC2 and its inhibitory activity against Streptococcus mutans

Background: Bacteriocin-like inhibitory substance (BLIS) demonstrates potent inhibitory effect against oral pathogens. To apply in oral care products, high purity of BLIS is required in view of the safety and effectiveness in the application. Therefore, aqueous two-phase systems (ATPS) are introduced for the recovery and purification of BLIS. Methods: The optimization of ionic liquid-based ATPS (ILATPS) for the purification of BLIS from Enterococcus faecium CC2 was performed using response surface methodology (RSM). The partially purified BLIS was then identified, and the inhibitory effect against Streptococcus mutans UKMCC1019 was assessed. Significant Findings: ILATPS with a pH of 7 at a temperature of 40 °C, comprising of 10 % (w/w) fermentation broth, 15 % (w/w) 1‑butyl‑3-methylimidazolium tetrafluoroborate, (Bmim)BF4, 12.5 % (w/w) sodium citrate and 3 % (w/w) sodium chloride, resulted in the optimum purification factor of 4.406, recovery yield of 67.213 % and partition coefficient of 4.852. The BLIS was identified as enterocin CC2, with a molecular size of approximately 5.5–6 kDa. Furthermore, the partially purified BLIS at a concentration of 1.00 mg/mL demonstrated effective growth inhibition against S. mutans UKMCC1019. The ILATPS was shown to be a potential technology to enhance the purity of BLIS from E. faecium CC2