RalA and RalB are members of the Ras family of small G proteins and are activated downstream of Ras via RalGEFs. The RalGEF-Ral axis represents one of the major effector pathways controlled by Ras and as such is an important pharmacological target. RalA and RalB are approximately 80% identical at the amino acid level; despite this, they have distinct roles both in normal cells and in the disease state. We have used our structure of RalB-RLIP76 to guide an analysis of Ral-effector interaction interfaces, creating panels of mutant proteins to probe the energetics of these interactions. The data provide a physical mechanism that underpins the effector selective mutations commonly employed to dissect Ral G protein function. Comparing the energetic landscape of the RalB-RLIP76 and RalB-Sec5 complexes reveals mutations in RalB that lead to differential binding of the two effector proteins. A panel of RLIP76 mutants was used to probe the interaction between RLIP76 and RalA and -B. Despite 100% sequence identity in the RalA and -B contact residues with RLIP76, differences still exist in the energetic profiles of the two complexes. Therefore, we have revealed properties that may account for some of the functional separation observed with RalA and RalB at the cellular level. Our mutations, in both the Ral isoforms and RLIP76, provide new tools that can be employed to parse the complex biology of Ral G protein signaling networks. The combination of these thermodynamic and structural data can also guide efforts to ablate RalA and -B activity with small molecules and peptides.
RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.
The cellular RNA content of mouse fibroblasts incubated with actinomycin decreases at a rate of about 1 to 1.5 per cent per hour, while DNA and protein content remain unchanged. This degradation affects nuclear and cytoplasmic RNA, ribosomal and soluble RNA. The breakdown products appear quantitatively in the acid-soluble fraction of the cells and the medium. Polynucleotides synthesized a short period (120 minutes) prior to exposure to actinomycin are degraded before those synthesized 8 to 12 hours previously.When mammalian cells are exposed for many hours to concentrations of actinomycin which completely block RNA synthesis, much of their cytochemically demonstrable RNA disappears, the ratio cellular DNA/RNA increases, and the morphology of their nucleoli is altered (I, 2). In order to obtain further information concerning the physiology of actinomycin-treated cells, we have studied the RNA metabolism of mouse fibroblasts (strain L-929) in culture, and the changes caused by actinomycin. The results show that cells in which RNA synthesis is suppressed by the antibiotic degrade a large percentage of their RNA. The process of depolymerization affects all cellular RNA species of the nucleus and of the cytoplasm. MATERIALS AND METHODSMouse fibroblasts were grown as monolayers in minimal Eagle's medium (3) supplemented with 5 per cent fetal bovine serum, or in spinner bottles as suspension cultures in Eagle's spinner medium (4) containing 10 per cent fetal bovine serum. Cells from monolayer cultures were harvested, after brief exposure to trypsin (0.4 mg/ml), by centrifugation in a refrigerated centrifuge, and washed twice with cold saline. All counts were performed in a hemocytometer. Cells were separated into nuclei and cytoplasm by the method of Harris (5), modified by extending the period of incubation in detergent to 20 minutes. The nuclei obtained by this method appeared free of cytoplasm when examined with the oil-immersion lens of a phase-contrast microscope; they contained 98 to 99 per cent of the cellular DNA as judged by their content of previously incorporated radioactive thymidine in acid-insoluble form. After separation of the nuclei by centrifugation at 300 g for 5 minutes, the snpernatant was centrifuged at 10,000 g for 15 minutes to remove mitochondria, and the ribosomes were isolated by centrifugation at 150,000 g for 180 minutes. Radioactive precursors were obtained from regular commercial sources. Radioactivity of cell fractions and extracts was measured in a scintillation counter.RNA was purified from whole cells, or from cell fractions, by a phenol method (6), in the presence of polyvinyl sulfate. After removal of phenol, the RNA was precipitated by 2 volumes of ethanol, redissolved and reprecipitated twice, and analyzed chromatographically on columns of methylated serum albuminkieselguhr (7), or in sucrose gradients (/3). The absorbancy (260 mp) and radioactivity of aliquots of fractions were measured. To measure nucleic acid and protein, cells were extracted and washed with perchloric ...
Background: Metabolic syndrome (MetS) is a multifactorial disease, whose main stay of prevention and management is life-style modification which is difficult to attain. Combination of herbs have proven more efficacious in multi-targeted diseases, as compared to individual herbs owing to the “effect enhancing and side-effect neutralizing” properties of herbs, which forms the basis of polyherbal therapies This led us to review literature on the efficacy of herbal combinations in MetS.Methods: Electronic search of literature was conducted by using Cinnahl, Pubmed central, Cochrane and Web of Science, whereas, Google scholar was used as secondary search tool. The key words used were “metabolic syndrome, herbal/poly herbal,” metabolic syndrome, clinical trial” and the timings were limited between 2005–2020.Results: After filtering and removing duplications by using PRISMA guidelines, search results were limited to 41 studies, out of which 24 studies were evaluated for combinations used in animal models and 15 in clinical trials related to metabolic syndrome. SPICE and SPIDER models were used to assess the clinical trials, whereas, a checklist and a qualitative and a semi-quantitative questionnaire was formulated to report the findings for animal based studies. Taxonomic classification of Poly herbal combinations used in animal and clinical studies was designed.Conclusion: With this study we have identified the potential polyherbal combinations along with a proposed method to validate animal studies through systematic qualitative and quantitative review. This will help researchers to study various herbal combinations in MetS, in the drug development process and will give a future direction to research on prevention and management of MetS through polyherbal combinations.
The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1 H, 15 N and 13 C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.
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