Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P,a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.     

Peters plus syndrome mutations affect the function and stability of human β1,3-glucosyltransferase

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes, have mutations in the gene encoding β1,3-glucosyltransferase [B3GLCT]). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.     

Elasticity and Rupture of a Multi-Domain Neural Cell Adhesion Molecule Complex

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of ∼0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.     

Computationally reconstructing cotranscriptional RNA folding from experimental data reveals rearrangement of non-native folding intermediates

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Section: bioprocessing and biological engineering gene fragment polymerization for increased yield of recombinant HIV fusion inhibitor enfuvirtide

Fuzeon (Enfuvirtide, T20) is the first fusion inhibitor approved by the FDA of the USA for the treatment of HIV/AIDS in combination with other anti-retroviral drugs. Enfuvirtide is a synthetic peptide that blocks the entry of HIV into healthy host CD₄ cells, which requires very high (90 mg twice daily) therapeutic doses. To increase the yield of Enfuvirtide, a gene polymerization strategy was introduced and recombinant T20 (rT20) was expressed in Escherichia coli as a five copy repeat polypeptide with a histidine-tag. The five copy rT20 was purified by Ni-affinity chromatography and cleaved to single rT20 units by cyanogen bromide. Finally, single rT20 units were purified by reversed phase chromatography giving a yield (400 mg/l) with a purity >95 %, which exhibited specific biological activity similar to Fuzeon.     

Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine

In eggplant (Solanum melongena L., cv. Pusa Purple Long), explantsfrom different regions of the leaf showed significant differencesfor embryogenic potential. Discs from the apical region of leavesyielded more somatic embryos than those from the basal region.Apical discs showed consistently higher polya-mine titres thanthe basal discs. Putrescine treatment promoted somatic embryogenesisand at 0.5 mM it caused a remarkable increase (c. 6-fold) ina number of somatic embryos, accompanied by an increased putrescinetitre. On the other hand, spermidine and spermine had no stimulatoryeffect on embryogenesis; rather they were inhibitory at higherconcentrations. All tested inhibitors of polyamine biosynthesissuch as difluoromethylarginine, difluoromethylomithine, methylglyoxalbis (guanylhydrazone) and bis (cyclohex-ylammonium) sulphatesignificantly inhibited somatic embryogenesis. Difluoromethylarginineblocked somatic embryogenesis by lowering endogenous polyaminecontents (particularly putrescine) and such inhibitory effectswere totally restored by exogenous putrescine (0.5 mM), concomitantwith the revival of endogenous PA concentrations. These resultsdemonstrate (i) a positive correlation between the spatial distributionof free and conjugated polyamines and the embryogenic capacityof an explant and (ii) putrescine caused the promotion of somaticembryogenesis, suggesting the intricate regulatory role of polyamines,specifically putrescine, in somatic embryogenesis in eggplant. Key words: Solanum melongena, somatic embryogenesis, position effect, polyamines, putrescine, polyamine biosynthesis inhibitors, difluoromethylarginine     

Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature—the calcium–myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins.     

Associations between Retinal Markers of Microvascular Disease and Cognitive Impairment in Newly Diagnosed Type 2 Diabetes Mellitus: A Case Control Study

Objective

To investigate associations between retinal microvascular changes and cognitive impairment in newly diagnosed type 2 diabetes mellitus.

Design

Case control study.

Setting

A primary care cohort with newly diagnosed type 2 diabetes mellitus.

Methods

For this analysis, we compared 69 cases with lowest decile scores (for the cohort) on the Modified Telephone Interview for Cognitive Status and 68 controls randomly selected from the remainder of the cohort. Retinal images were rated and the following measures compared between cases and controls: retinal vessel calibre, arterio-venous ratio, retinal fractal dimension, and simple and curvature retinal vessel tortuosity.

Results

Total and venular (but not arteriolar) simple retinal vessel tortuosity levels were significantly higher in cases than controls (t = 2.45, p = 0.015; t = 2.53, p = 0.013 respectively). The associations persisted after adjustment for demographic factors, retinopathy, neuropathy, obesity and blood pressure. There were no other significant differences between cases and controls in retinal measures.

Conclusions

A novel association was found between higher venular tortuosity and cognitive impairment in newly diagnosed type 2 diabetes mellitus. This might be accounted for by factors such as hypoxia, thrombus formation, increased vasoendothelial growth factor release and inflammation affecting both the visible retinal and the unobserved cerebral microvasculature.     

A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.