Cysteine Mutants Research Articles

Next-generation sequencing panel analysis in 24 Chinese patients with congenital ectopia lentis.

Congenital ectopia lentis (CEL) is a rare but serious disease. We use next-generation sequencing to detect genes associated with lens abnormalities in 24 patients with bilateral CEL and search for pathogenic genes and mutation sites. A total of 24 patients diagnosed with CEL from January 2019 to November 2019 were enrolled in this study, and their clinical data were collected and genome-wide deoxyribonucleic acid was extracted from peripheral venous blood. Targeted gene capture technology was used to obtain 188 exons of lens abnormality-related genes, which were sequenced using a high-throughput method. The mutation sites were determined through data analysis and verified by the Sanger method. According to the data from previous studies, the association between the genotype and clinical phenotype was analysed. Of the 24 patients, 23 had mutations in the fibrillin-1 (FBN1) gene, and 20 were diagnosed with Marfan syndrome. The 23 cases of FBN1 mutations were all heterozygous mutations, including 17 missense mutations, 3 splicing variants, 2 exon deletion mutations, 1 codon mutation, and 9 new mutations. A total of 17 mutations were located in the calcium-binding epidermal growth factor domain, including 16 mutations that contained missense mutations of cysteine. In addition, a heterozygous mutation of the gap junction protein alpha 8 (GJA8) gene was detected in one patient. In this study, we identified 23 FBN1 gene mutations and 1 GJA8 gene mutation in 24 patients with CEL. Of these, 9 new FBN1 mutations and 14 known mutations were found. The results expanded the mutation spectrum of the FBN1 gene, suggesting that FBN1 mutation may be the main cause of CEL in Chinese patients.

Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains.

Botrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein–protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein–protein interactions occurring between the core elements of the B. cinerea circadian clock.

Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

Dissecting molecular mechanisms underlying T cell co-potentiation when targeting the TCR/CD3 complex with anti-CD3 Fab fragments

Abstract A cognate antigen engagement of TCR induces a conformational change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens that do not active T cells. Anti-CD3 monovalent Fab fragments bound to the CD3 complex mimic CD3Δc, producing T cell “co-potentiation” when associated with TCRs bound to weak antigens that enhances T cell activation, neither stimulating non-engaged T cells, nor interrupting T cell responses to strong antigens. Previously, we showed T cell co-potentiation can be exploited as a novel immunotherapeutic principle by reducing melanoma burden in B6 mice treated with anti-CD3 Fabs. TCR signaling initiation requires CD3Δc for the exposure of a conserved sequence in the cytosolic domain of CD3ɛ rich in prolines (PRS) that enables the recruitment of adaptor protein Nck. The mutation of cysteines in the CXXC motif at the extracellular domain of CD3ɛ inhibits the conformational change across the plasma membrane that results on PRS accessibility. Here, using two strains of B6 mice that express mutations in either the CXXC or the PRS motifs in CD3ɛ, we study the potential contribution of each motif in T cell co-potentiation. Our results from these studies favor the involvement of the CXXC and PRS motifs in the molecular mechanism supporting the co-potentiation of T cell activation by anti-CD3 Fab fragments. Supported by MU Start-up funds and NIH grants: NCI, U01 CA244314; NIAID, R01 AI097187

Abstract 193: Apolipoprotein A-I Rotamers Determine The Size And Cholesterol Efflux Capacity Of High-Density Lipoprotein

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers in vivo , we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient ( Apoa1 -/- ) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P<0.0001) than those of LL5/4 mice. Importantly, mice expressing different APOA1 rotamers had markedly different HDL size patterns: HDL in LL5/4 hAPOA1 mice consisted mainly of extra-small HDL (7.8 nm in diameter), consistent with our finding that the LL5/4 rotamer had a low efficiency in activating LCAT. HDL in WT and LL5/5 mice had the same size distribution with one major peak at 10 nm. LC-MS/MS analysis identified 48 HDL proteins. One-third of the proteins (including APOA2, APOC1, APOC3 and LCAT) differed significantly in relative abundance in the different rotamers. Importantly, on a per particle basis, HDL from LL5/4 mice demonstrated three times higher ABCA1 cholesterol efflux capacity than HDL from WT and LL5/5 mice. Our observations strongly suggest that APOA1 rotamers play distinct roles in HDL metabolism, raising the possibility that differences in APOA1 rotamer distribution alter the cardioprotective functions of HDL.

Measuring Cytotoxicity of the Designed Antimicrobial Peptides DesHDAP1 and DesHDAP1‐C on Human Embryonic Kidney Cells

Antimicrobial peptides (AMPs) have emerged as a promising alternative to traditional antibiotics, particularly in light of multi‐antibiotic resistant strains of bacteria. Histone derived AMPs (HDAPs) are an integral part of this toolset, with multiple mechanisms of action, strong antimicrobial effects, and protein design potential. Our lab has previously designed two robust antimicrobial peptides: DesHDAP1 and the subsequent DesHDAP1‐C, which has an additional cysteine residue to facilitate conjugation to other molecules for fluorescence imaging or attachment to active moieties. Previous work demonstrated that the cysteine mutation did not alter antibacterial activity of the DesHDAP1 peptide. However, it is also critical to determine whether this change affects potential toxicity against eukaryotic cells. Using dye‐based assays, we quantified cellular metabolism of human embryonic kidney cells as a proxy for cell viability. By optimizing this process and the data analysis, I was able to compare cytotoxicity of the two designed peptides to that of conventional antibiotics. This approach can be applied to other designed peptides, providing a blueprint for future toxicity studies on eukaryotic cells.

Treatment with IFB-088 Improves Neuropathy in CMT1A and CMT1B Mice

Charcot-Marie-Tooth disease type 1A (CMT1A), caused by duplication of the peripheral myelin protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene, are the two most common forms of demyelinating CMT (CMT1), and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modelled by MpzR98C/ + mice that also show ER stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A, we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analysed by behavioural, neurophysiological, morphological and biochemical measures. Both MpzR98C/ + and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild-type values. Taken together, our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in amyotrophic lateral sclerosis and multiple sclerosis animal models, these data demonstrate its potential in managing UPR and ER stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases.Graphical (Left panel) the accumulation of overexpressed PMP22 or misfolded mutant P0 in the Schwann cell endoplasmic reticulum (ER) leads to overwhelming of the degradative capacity, activation of ER-stress mechanisms, and myelination impairment. (Right panel) by prolonging eIF2α phosphorylation, IFB-088 reduces the amount of newly synthesized proteins entering the ER, allowing the protein quality control systems to better cope with the unfolded/misfolded protein and allowing myelination to progress.

Discovery of AZD4625, a Covalent Allosteric Inhibitor of the Mutant GTPase KRASG12C.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Cysteine-Based Mimic of Arginylation Reproduces Neuroprotective Effects of the Authentic Post-Translational Modification on α-Synuclein.

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.

HECTD3 promotes gastric cancer progression by mediating the polyubiquitination of c-MYC

The E3 ubiquitin ligase HECTD3 is homologous with the E6 related protein carboxyl terminus, which plays a vital role in biological modification, including immunoreactivity, drug resistance and apoptosis. Current research indicates that HECTD3 promotes the malignant proliferation of multiple tumors and increases drug tolerance. Our study primarily explored the important function and effects of HECTD3 in gastric cancer. Here, we discovered that HECTD3 is abnormally activated in gastric cancer, and the clinical prognosis database suggested that HECTD3 was strongly expressed in gastric cancer. Depletion of HECTD3 restrained the proliferative and clone abilities of cells and induced the apoptosis of gastric cancer cells. Mechanistically, our findings revealed that interaction between HECTD3 and c-MYC, and that the DOC domain of HECTD3 interacted with the CP and bHLHZ domains of c-MYC. Furthermore, we discovered that HECTD3 mediates K29-linked polyubiquitination of c-MYC. Then, our research indicated that cysteine mutation at amino acid 823 (ubiquitinase active site) of HECTD3 reduces the polyubiquitination of c-MYC. Our experimental results reveal that HECTD3 facilitates the malignant proliferation of gastric cancer by mediating K29 site-linked polyubiquitination of c-MYC. HECTD3 might become a curative marker.

Enhancing the thermostability of transglutaminase from Streptomyces mobaraensis based on the rational design of a disulfide bond

Transglutaminase (TGase), a transferase, is widely adopted in the food industry and other biological fields due to its unique characteristics of modifying proteins by intra- or intermolecular cross-linking. However, obtaining a mutant TGase that is highly thermostable and active would significantly aid in food processing. Therefore, this study sought to improve the thermostability of TGase by introducing an artificial disulfide bridge through a structure-based rational enzyme engineering approach. After the rational screening, six disulfide mutants (E139C/G143C, R146C/E149C, A182C/N195C, L200C/R208C, T223C/F226C, and E139C/G143C+L200C/R208C) of the transglutaminase gene from Streptomyces mobaraensis (Sm-TGase) were selected and constructed by rationally designed mutations in cysteine. Of them, a mutant (E139C/G143C) with enhanced thermostability was selected and characterized for further analysis. The results indicated that the mutant E139C/G143C had a similar specific activity, optimal temperature, and pH but a lower Km and higher Vmax than the wild-type. Its half-life (t1/2) at 55 °C was 10.7 min, which was 1.69-fold higher than the wild-type, while its melting temperature (Tm) was 3.52 °C higher than the wild-type. These results proved that the introduction of disulfide bonds into TGase by rational design could be an effective approach to improve the thermostability of TGase and other food enzymes for food processing.

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

Comparison between reduction rates of wild-type and resolving cysteine mutants of yeast typical 2-Cys Prx by thioredoxin

Comparison between reduction rates of wild-type and resolving cysteine mutants of yeast typical 2-Cys Prx by thioredoxin

A Palette of Fluorescent Aβ42 Peptides Labelled at a Range of Surface-Exposed Sites.

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of A42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled A42 peptides that can be used to gain understanding of the complex mechanisms of A42 self-assembly and help to develop a more targeted approach to cure the disease.

Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Rare by Natural Selection: Disulfide-Bonded Supramolecular Antimicrobial Peptides

Human LL-3717-29 is an antimicrobial peptide forming thermostable supramolecular fibrils that surround bacterial cells. The crystal structure of LL-3717-29 bearing an I24C substitution of most buried position in the fibril revealed disulfide-bonded dimers that further assembled into a fibrillar structure of densely packed helices. We further demonstrated the position-dependent controllable antibacterial activity of LL-3717-29 I24C and other cysteine mutants, mediated by regulation of intermolecular disulfide bonds and their role in the formation of supramolecular structures. The morphology of the fibrils and their antibacterial mechanism of action might be dependent on their interactions with specific bacteria. The significant effect of disulfide bonds on the assembly into supramolecular structures and their sensitivity to reducing/oxidizing conditions may explain why short helical antimicrobial peptides with a single cysteine and an odd number of cysteines are selected against in nature.

Voltage-Dependent Anion Selective Channel 3: Unraveling Structural and Functional Features of the Least Known Porin Isoform.

Voltage-dependent anion-selective channels (VDAC) are pore-forming proteins located in the outer mitochondrial membrane. Three isoforms are encoded by separate genes in mammals (VDAC1-3). These proteins play a crucial role in the cell, forming the primary interface between mitochondrial and cellular metabolisms. Research on the role of VDACs in the cell is a rapidly growing field, but the function of VDAC3 remains elusive. The high-sequence similarity between isoforms suggests a similar pore-forming structure. Electrophysiological analyzes revealed that VDAC3 works as a channel; however, its gating and regulation remain debated. A comparison between VDAC3 and VDAC1-2 underlines the presence of a higher number of cysteines in both isoforms 2 and 3. Recent mass spectrometry data demonstrated that the redox state of VDAC3 cysteines is evolutionarily conserved. Accordingly, these residues were always detected as totally reduced or partially oxidized, thus susceptible to disulfide exchange. The deletion of selected cysteines significantly influences the function of the channel. Some cysteine mutants of VDAC3 exhibited distinct kinetic behavior, conductance values and voltage dependence, suggesting that channel activity can be modulated by cysteine reduction/oxidation. These properties point to VDAC3 as a possible marker of redox signaling in the mitochondrial intermembrane space. Here, we summarize our current knowledge about VDAC3 predicted structure, physiological role and regulation, and possible future directions in this research field.

Establishing the mutational effect on the binding susceptibility of AMG510 to KRAS switch II binding pocket: Computational insights

After decades of leaving patients with limited treatment options due to the ‘undruggability’ of Kirsten rat sarcoma, the kernel was finally cracked with the discovery of Sotorasib. This novel drug binds to a small pocket on the switch II of Kirsten rat sarcoma by exploiting the mutation that occurs at codon 12 wherein glycine is replaced by cysteine. However, this pocket is not only prone to cysteine mutation but other mutations occur as well including at codon 13. These mutations have been reported to drive cancer in the lungs, colorectal and in solid tumors with varying degree of expressions. Sotorasib is therefore only effective in a small group of patients especially those expressing cysteine at codon 12, reminding drug hunters of the unfinished work. This study employs computational techniques to understand the susceptibility of the mutated binding pocket to the binding of Sotorasib. It was revealed that the binding affinity of Sotorasib to other mutations aside Cysteine was significantly affected via the total free binding energies presented by the mutated complexes. Furthermore, the quantum of energy contributed by the mutated residues except cysteine was significantly reduced suggesting the binding of Sotorasib is enhanced in only cysteine mutated Kirsten rat sarcoma. Residual interaction network showed Sotorasib in complex with the cysteine mutated protein presented the highest degree centrality, shortest path betweenness, shortest path centrality and shortest path degree indicating Sotorasib controls more information flow within the cysteine mutated protein than in other mutations. Insight unraveled here will provide aid in the development of pan Kirsten rat sarcoma inhibitory small molecules.

Molecular insights on the conformational dynamics of a P76C mutant of human cytochrome c and the enhancement on its peroxidase activity

In apoptotic pathway, the interaction of Cytochrome c (Cytc) with cardiolipin in vivo is a key process to induce peroxidase activity of Cytc and trigger the release of Cytc in the inner mitochondria into cytosol. The peroxidase active form of Cytc occurs due to local conformational changes that support the opening of the heme crevice and the loss of an axial ligand between Met80 and heme Fe. Structural adjustments at the Ω-loop segments of Cytc are required for such process. To study the role of the distal Ω-loop segments comprising residues 71–85 in human Cytc (hCytc), we investigated a cysteine mutation at Pro76, one of the highly conserved residues in this loop. The effect of P76C mutant was explored by the combination of experimental characterizations and molecular dynamics (MD) simulations. The peroxidase activity of the P76C mutant was found to be significantly increased by ∼13 folds relative to the wild type. Experimental data on global denaturation, alkaline transition, heme bleaching, and spin-labeling Electron Spin Resonance were in good agreement with the enhancement of peroxidase activity. The MD results of hCytc in the hexacoordinate form suggest the important changes in P76C mutant occurred due to the unfolding at the central Ω-loop (residues 40–57), and the weakening of H-bond between Tyr67 and Met80. Whereas the experimental data implied that the P76C mutant tend to be in equilibrium between the pentacoordinate and hexacoordinate forms, the MD and experimental information are complementary and were used to support the mechanisms of peroxidase active form of hCytc.

Targeting KRAS G12C with Covalent Inhibitors

KRAS is the most frequently mutated oncogene in cancer. Following numerous attempts to inhibit KRAS spanning multiple decades, recent efforts aimed at covalently targeting the mutant cysteine of KRAS G12C have yielded very encouraging results. Indeed, one such molecule, sotorasib, has already received accelerated US Food and Drug Administration approval with phase III clinical trials currently underway. A second molecule, adagrasib, has also progressed to phase III, and several others have entered early-phase clinical trials. The success of these efforts has inspired an array of novel approaches targeting KRAS, with some reporting extension to the two most common oncogenic KRAS mutations, G12V and G12D.