Cysteine Mutants Research Articles

Effect of cysteine mutation at Ca2+ coordinating residues to the autolysis, folding and hydrophobicity of full length and mature Rand protease: molecular dynamics simulation and essential dynamics

Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic solvent and broad in specificity. Many structures of homologous protein solved by X-ray crystallography and NMR have been deposited to Protein Data Bank (PDB) which allowed this study to rely on structure prediction by deep learning to build three-dimensional (3D) structure of full length and mature Rand protease (flRP and mRP). In silico cysteine mutation to 7 predicted high affinity Ca2+ coordinating residues were introduced, and the mutants were subjected to molecular dynamics simulation to study its effect on flRP and mRP. MD simulation showed a marked increase in flexibility of the pro-peptide segment indicating the impact of single cysteine substitution at high affinity Ca2+ coordinating residues to autolysis of flRP. MD simulation for mRP reaffirmed the role of Ca2+ coordinating sites in providing stability to Rand protease. In addition, these residues also affect the autolysis, folding and hydrophobicity of RP. Essential dynamics observed large contribution of the first few eigenvectors of flRP, mRP and their high affinity Ca2+ coordinating residues mutants to the TMSF values which indicates that these values account for a large portion of the overall atomic fluctuations. These results have given a more comprehensive understanding on the role of cysteine substituted Ca2+ coordinating surface loop to the structure of flRP and mRP which are important in contributing to the structural stability of subtilisin. Communicated by Ramaswamy H. Sarma

Precision fermentation as a route to modify β-lactoglobulin structure through substitution of specific cysteine residues

Precision fermentation is a promising route to develop recombinant proteins with improved functionality. Protein functionality is highly influenced by protein structure, which can be significantly affected by conformational restraints induced by cysteines. Here, we study the impact of individual cysteines on β-lactoglobulin structure, by stepwise substituting them with alanine. Structural characterisation was performed from secondary to quaternary level. The findings demonstrated that the free Cys121 played no role in the folding and dimerisation of BLG. The β-barrel usually includes one disulphide bond (Cys106–Cys119), which was found to be crucial for its formation. The usually exposed disulphide bond (Cys66–Cys160) played no major role in folding of the β-barrel, but mainly modulated its accessibility and mediated dimerisation. Thereby, cysteine mutations can be used as a tool to modify protein structures. These different structures can now be used to further understand the impact on protein behaviour and their functionality.

Recent Developments, Challenges and Opportunities in Targeting the KRAS Pathway

Abstract: KRAS is a highly mutated gene among cancers, especially in the lung, pancreatic and colorectal cancers (CRC). Despite extensive efforts in the past three decades, KRAS remains undruggable. But lately, inhibitors selectively binding to cysteine mutant at 12th position (G12C) are in clinical trials as a single agent and in combination with other drugs. FDA approved LUMAKRAS (Sotorasib, AMG510) for metastatic NSCLC, while Adagrasib (MRTX849) is under clinical trials, and it is showing good safety and efficacy in pancreatic and GI tumors. Both these molecules demonstrated better response in NSCLC but have less efficacy in colorectal and other solid cancers. Among non-G12C KRAS mutant cancers, promising data are emerging from G12D inhibition. This review covers the status of KRAS G12C, non- G12C inhibitors, and discusses different nodal proteins in the RAS signaling pathway, mechanism of resistance in targeted therapy and combination approaches. Ongoing clinical trials with G12C inhibitors have promising results. However, a combination of KRAS inhibition with other inhibitors of signaling components in vertical axis (SHP2, SOS1, MEK, PI3K/AKT and EGFR inhibitors) might be beneficial for durable clinical response. Sotorasib in combination with MEK inhibitor showed promising results in preclinical xenograft and PDx models of KRAS mutated cancers. Although at a preclinical stage, other approaches like Tri complex inhibitor, KRAS (ON) inhibitors, and non-G12C inhibitors are of significant interest in KRAS drug discovery. Multiple targeted approaches are being explored based on tumor specific gene expression profiles considering dynamic nature of KRAS activity and heterogeneity of hard to treat tumors.

Ocular, cardiovascular, and genetic characteristics and their associations in children with Marfan syndrome and related fibrillinopathies.

Congenital ectopia lentis (CEL) and heart abnormalities are common clinical symptoms in patients with Marfan syndrome (MFS) and related fibrillinopathies, which is caused by mutations in fibrillin-1 (FBN1) gene. This study aims to explore the ocular and cardiovascular characteristics and their association with genotype in children with MFS and related fibrillinopathies. Seventy-nine children diagnosed with CEL and with FBN1 mutations confirmed via whole-exome sequencing were included for genotypes and phenotypes analysis. The axial length (AL), corneal curvature, and refractive status were included for ocular phenotypes analysis. The cardiovascular examination was performed by echocardiography, and aortic root Z score was calculated to evaluate the severity of aortic dilatation. The heart disorders were classified as aortic root dilatation, valvular disorders, and others. Both the ocular and cardiac manifestations were collected for comprehensive analysis and compared among patients with different genotypes, including the mutation involving cysteine substitution or mutation in different regions. In CEL children with FBN1 mutations, 77.2% patients could be diagnosed as MFS. It was observed that children with mutations in exons 22-42 had significant higher aortic root Z score (P = 0.003) and higher incidence of cardiovascular disorders (P = 0.004). Additionally, children with cysteine substitution mutations had significant higher aortic root Z score (P = 0.011), and the aortic root Z score was positively associated with axial length (AL) in children under 6years old (P = 0.035). Those with long AL (≥ 26mm) had significant higher incidence of valve disorders (P = 0.023). In addition, nearly half the children with CEL (46.8%) were diagnosed with cardiovascular disease for the first time. CEL children with FBN1 mutations involving cysteine substitution or mutations in exons 22-42 or with long AL had higher risks of severe cardiovascular complications. Knowing the phenotype may help in anticipating severe cardiovascular disease in CEL patients.

Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

E3 ubiquitin ligase HECTD3 is a tumor suppressor and mediates the polyubiquitination of SLC7A11 to promote ferroptosis in colon cancer

Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) has been reported to play an essential role in biological processes, including drug resistance, metastasis or apoptosis. However, the relationships between HECTD3 and Colorectal cancer (CRC) remain to be unclear. In this study, we discovered that HECTD3 expressed lowly in CRC compared with normal tissues and patients with low HECTD3 suffered from poorer survival outcomes relative to those with high HECTD3 levels. HECTD3 inhibition could significantly enhance proliferative, clone abilities and self-renewal capacities of CRC cells in vitro and in vivo. Mechanistically, our findings revealed that HECTD3 had endogenous interactions with SLC7A11 proteins. HECTD3 promoted the polyubiquitination of SLC7A11 to trigger the degradation of SLC7A11 proteins. Targeting HECTD3 could notably prolong the half-life period of SLC7A11 proteins, thereby promoting its stability. However, the cysteine mutation at amino acid 823 (ubiquitinase active site) of HECTD3 impaired the polyubiquitination of SLC7A11. HECTD3 deficiency depended on accumulated SLC7A11 proteins to accelerate malignant progression of CRC in vitro and in vivo. Thus, HECTD3 could suppress SLC7A11 levels to attenuate the SLC7A11-mediated cystine uptake, leading to enhanced CRC ferroptosis. SLC7A11 inhibition through polyubiquitination by HECTD3 increased ferroptosis, thereby inhibiting CRC tumor growth. Taken together, these results showed that HECTD3 controlled the stability of SLC7A11 and uncovered the function of HECTD3/SLC7A11 axis in regulating CRC progression.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

Structural changes in NOTCH3 induced by CADASIL mutations: Role of cysteine and non-cysteine alterations

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that results from mutations in NOTCH3. How mutations in NOTCH3 ultimately result in disease is not clear, although there is a predilection for mutations to alter the number of cysteines of the gene product, supporting a model in which alterations of conserved disulfide bonds of NOTCH3 drives the disease process. We have found that recombinant proteins with CADASIL NOTCH3 EGF domains 1 to 3 fused to the C terminus of Fc are distinguished from wildtype proteins by slowed mobility in nonreducing gels. We use this gel mobility shift assay to define the effects of mutations in the first three EGF-like domains of NOTCH3 in 167 unique recombinant protein constructs. This assay permits a readout on NOTCH3 protein mobility that indicates that (1) any loss of cysteine mutation in the first three EGF motifs results in structural abnormalities; (2) for loss of cysteine mutants, the mutant amino acid residue plays a minimal role; (3) the majority of changes that result in a new cysteine are poorly tolerated; (4) at residue 75, only cysteine, proline, and glycine induce structural shifts; (5) specific second mutations in conserved cysteines suppress the impact of loss of cysteine CADASIL mutations. These studies support the importance of NOTCH3 cysteines and disulfide bonds in maintaining normal protein structure. Double mutant analysis suggests that suppression of protein abnormalities can be achieved through modification of cysteine reactivity, a potential therapeutic strategy.

Exploring Redox Modulation of Plant UDP-Glucose Pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

A Point Mutation at C151 of Keap1 of Mice Abrogates NRF2 Signaling, Cytoprotection in Vitro, and Hepatoprotection in Vivo by Bardoxolone Methyl (CDDO-Me).

Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid in late-stage clinical development for the treatment of patients with diabetic kidney disease. Preclinical studies in rodents demonstrate the efficacy of triterpenoids against carcinogenesis and other diseases, including renal ischemia-reperfusion injury, hyperoxia-induced acute lung injury, and immune hepatitis. Genetic disruption of Nrf2 abrogates protection by triterpenoids, suggesting that induction of the NRF2 pathway may drive this protection. Herein, we examined the effect of a point mutation (C151S) in KEAP1, a repressor of NRF2 signaling, at cysteine 151 in mouse embryo fibroblasts and mouse liver. Induction of target gene transcripts and enzyme activity by CDDO-Me was lost in C151S mutant fibroblasts compared with wild-type. Protection against menadione toxicity was also nullified in the mutant fibroblasts. In mouse liver, CDDO-Me evoked the nuclear translocation of NRF2, followed by increased transcript and activity levels of a prototypic target gene, Nqo1, in wild-type, but not C151S mutant, mice. To test the role of KEAP1 Cys151 in governing the broader pharmacodynamic action of CDDO-Me, wild-type and C151S mutant mice were challenged with concanavalin A to induce immune hepatitis. Strong protection was seen in wild-type but not C151S mutant mice. RNA-seq analysis of mouse liver from wild-type, C151S mutant, and Nrf2-knockout mice revealed a vigorous response of the NRF2 transcriptome in wild-type, but in neither C151S mutant nor Nrf2-knockout, mice. Activation of "off-target" pathways by CDDO were not observed. These data highlight the singular importance of the KEAP1 cysteine 151 sensor for activation of NRF2 signaling by CDDO-Me. SIGNIFICANCE STATEMENT: KEAP1 serves as a key sensor for induction of the cytoprotective signaling pathway driven by the transcription factor NRF2. Mutation of a single cysteine (C151) in KEAP1 abrogates the induction of NRF2 signaling and its downstream cytoprotective actions in vitro and in vivo by bardoxolone methyl (CDDO-Me), a drug in late-stage clinical development. Further, at these bioeffective concentrations/doses, activation of "off-target" pathways by CDDO-Me are not observed, highlighting the singular importance of NRF2 in its mode of action.

Varied T cell repertoire bearing intact TCR/CD3 complexes required to observe maximal therapeutic anti-tumor effects of CD3 Fab fragments when binding to T cells

Abstract A cognate antigen engagement of TCR induces a conformation change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens, like many tumor-associated-antigens (TAAs), which do not active T cells. Anti-CD3 monovalent Fab fragments (Mono-Fabs) 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 Mono-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 of CD3ɛ extracellular domain inhibits the transmission of TCR signaling across transmembrane that defects CD3Δc and PRS accessibility. Here, using the B6 syngeneic melanoma cell lines Yumm 1.7 and Yummer1.7, which differ in the number of somatic mutations they carry, in B6 mouse strains either wild or mutated in either the CXXC or the PRS motifs in CD3ɛ, we have established the need of tumor cells to carry potential tumor neoantigens, and a more varied repertoire of T cells responding to the tumor with intact TCR/CD3 complexes to observe full anti-tumor effects by anti-CD3 Mono-Fab treatments. MU Start-up funds NIH, NCI, U01 CA244314 NIH, NIAID, R01 AI097187

Abstract IA08: Direct covalent inhibitors of mutant K-Ras

Abstract Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. I will discuss the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras G12C. These compounds rely on the mutant cysteine for binding and therefore do not affect the wild type protein (WT). New covalent molecules targeting K-Ras G12S and G12D are currently under development and will be discussed. I will also discuss reactivation of mutant p53 (Y220C) to leverage the natural anti-tumor effects of tumor suppressors. Citation Format: Kevan M. Shokat. Direct covalent inhibitors of mutant K-Ras [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA08.

Abstract 4442: Dual benefit of MALT1 blockade in glioblastoma

Abstract Background: Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor. Despite advances in treatment modalities, GBM remains largely incurable and new treatment approaches are desperately needed. An innovative strategy for cancer therapy is to combine the inhibition of cancer cell-intrinsic oncogenic signaling with cancer cell-extrinsic immunological activation of the tumor microenvironment (TME). MALT1 represents a candidate target whose inhibition may provide this dual benefit. Originally identified as a proto-oncoprotein in lymphoma, MALT1 has recently also been implicated in multiple solid tumors, including GBM, and significant effort has been made to develop MALT1 protease inhibitors as anti-cancer agents. Interestingly, blockade of MALT1 protease has also been shown to selectively reprogram tumor infiltrating Treg cells into antitumor effector cells. Based on these observations, we hypothesized that in GBM, MALT1 acts both within tumor cells and within cells of the TME to promote tumorigenesis and MALT1 inhibitor treatment could thus have dual benefit. Methods/Results: We assembled a panel of mouse and human GBM cell lines and performed western-blot analysis to show that these cells demonstrate constitutive MALT1 expression and proteolytic activity. MALT1 blockade in GBM cells, using siRNA or MALT1 protease inhibitors, reduces viability and clonogenic potential, suggesting that inhibition of MALT1 could impair tumor cell survival. Single-cell RNAseq data from public datasets demonstrated that myeloid cells have the highest MALT1 expression in the GBM TME. Our data indicate that co-culture of macrophages with GBM cells induces MALT1-NF-κB activation within the macrophages and this is associated with polarization towards an M2-like immunosuppressive phenotype. Additionally, treating primary human or mouse macrophages with MALT1-protease inhibitor prevents GBM-induced M2-polarization, increases M1-polarization and enhances GBM cell killing. In a syngeneic orthotopic mouse model, GBM tumor growth is impaired, and survival is extended when tumor cells are implanted into MALT1-protease dead (PD) host mice (mice which harbor a point mutation in the MALT1-protease catalytic cysteine) as compared to when implanted into wild-type host mice. MALT1-PD mice demonstrate a less immunosuppressive GBM TME with increased M1-macrophages and decreased M2-macrophages, MDSCs and Tregs. Taken together, our findings suggest that inhibiting MALT1 protease in TME cells promotes anti-tumor immunity against brain tumors. Conclusions: Our studies suggest MALT1 protease as a new therapeutic target in GBM whose inhibition could have dual benefit via both direct effect on tumor cells and by reversing the immunosuppressive TME. Citation Format: Juliana Hofstatter Azambuja, Saigopalakrishna S. Yerneni, Gabriela N. Debom, Lisa M. Maurer, Hannah E. Butterfield, Linda R. Klei, Gary Kohanbash, Peter C. Lucas, Linda M. McAllister-Lucas. Dual benefit of MALT1 blockade in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4442.

Segmentation of incident lacunes during the course of ischemic cerebral small vessel diseases.

Lacunes represent key imaging markers of cerebral small vessel diseases (cSVDs). During their progression, incident lacunes are related to stroke manifestations and contribute to progressive cognitive and/or motor decline. Assessing new lesions has become crucial but remains time-consuming and error-prone, even for an expert. We, thus, sought to develop and validate an automatic segmentation method of incident lacunes in CADASIL caused by cysteine mutation in the EGFr domains of the NOTCH3 gene, a severe and progressive monogenic form of cSVD. Incident lacunes were identified based on difference maps of 3D T1-weighted MRIs obtained at the baseline and 2 years later. These maps were thresholded using clustering analysis and compared with results obtained by expert visual analysis, which is considered the gold standard approach. The median number of lacunes at the baseline in 30 randomly selected patients was 7 (IQR = [2, 11]). The median number of incident lacunes was 2 (IQR = [0, 3]) using the automatic method (mean time-processing: 25 s/patient) and 0.5 (IQR = [0, 2]) using the standard visual approach (mean time-processing: 8 min/patient). The complementary analysis of segmentation results is enabled to quickly remove false positives detected in specific locations and to identify true incident lesions not previously detected by the standard analysis (2 min/case). A combined approach based on automatic segmentation of incident lacunes followed by quick corrections of false positives allowed to reach high individual sensitivity (median at 0.66, IQR = [0.21, 1.00]) and global specificity scores (0.80). The automatic segmentation of incident lacunes followed by quick corrections of false positives appears promising for properly and rapidly quantifying incident lacunes in large cohorts of cSVDs.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

UBE2E2 enhances Snail-mediated epithelial-mesenchymal transition and Nrf2-mediated antioxidant activity in ovarian cancer

Dissemination of ovarian cancer (OvCa) cells can lead to inoperable metastatic lesions in the bowel and omentum, which have a poor prognosis despite surgical and chemotherapeutical options. A better understanding of the mechanisms underlying metastasis is urgently needed. In this study, bioinformatics analyses revealed that UBE2E2, a less-studied ubiquitin (Ub)-conjugating enzyme (E2), was upregulated in OvCa and was associated with poor prognosis. Subsequently, we performed western blot analysis and IHC staining with 88 OvCa and 26 normal ovarian tissue samples, which further confirmed that UBE2E2 protein is highly expressed in OvCa tissue but weakly expressed in normal tissue. Furthermore, the silencing of UBE2E2 blocked OvCa cell migration, epithelial-mesenchymal transition (EMT) and metastasis in vitro, whereas UBE2E2 overexpression exerted the opposite effects. Mechanistically, UBE2E2 promoted p62 accumulation and increased the activity of the Nrf2-antioxidant response element (ARE) system, which ultimately activated the Snail signaling pathway by inhibiting the ubiquitin-mediated degradation of Snail. Additionally, co-IP and immunofluorescence demonstrated that a direct interaction exists between UBE2E2 and Nrf2, and the N-terminal of UBE2E2 (residues 1-52) is required and sufficient for its interaction with Nrf2 protein. Mutations in the active site cysteine (Cys139) impaired both the function and cellular distribution of UBE2E2. More importantly, the deletion of UBE2E2 reduced tumorigenicity and metastasis in xenograft OvCa mouse models. Taken together, our findings reveal the role of the UBE2E2-Nrf2-p62-Snail signaling axis in OvCa and thus provides novel therapeutic targets for the prevention of OvCa metastasis.

Intersubunit cross-linking is the key mechanism of cardiac ryanodine receptor dysfunction during oxidative stress: The role of cysteines 1078 and 2991.

The type 2 ryanodine receptor (RyR2) plays a key role in regulating cardiac intracellular Ca2+. We have previously shown that oxidative stress can regulate RyR2 by promoting the formation of disulfide bonds between neighboring RyR2 subunits (intersubunit cross-linking). However, the functional significance of this redox modification and cysteines involved in cross-linking remain unknown. Here, we used site-directed mutagenesis to replace cysteines 1078 and 2991 with serines in human RyR2 (hRyR2). Obtained constructs were expressed in HEK293 cells and treated with diamide to induce oxidative stress. Western blot analysis revealed that both mutations substantially protected against hRyR2 cross-linking. Analysis of the ER lumen [Ca2+] ([Ca2+]ER) dynamics with the ER Ca2+ sensor R-CEPIA1er in HEK293 cells expressing hRyR2 revealed that the mutation of either 1078 or 2991 cysteines prevented the depletion of ER Ca2+ load caused by RyR2 oxidation (by >40%). The simultaneous mutation of both cysteines produced the similar protection against hRyR2 cross-linking and [Ca2+]ER depletion, indicating a common mechanism of redox regulation involving both residues. Given the structural localization of both cysteines at the neighboring RyR2 subunits, we suggest that they form an intersubunit disulfide bond during channel oxidation, followed by the formation of the pathologically “leaky” channel. Although the N-terminal region (NTR) of RyR2 plays an important role in the channel's tetramer formation and functional regulation, the individual cysteines in this region are not directly involved in hRyR2 cross-linking. However, the structural integrity of the NTR has an allosteric effect on hRyR2 complex re-arrangements required for the channel's cross-linking. In conclusion, the results of this study revealed two critical cysteines within hRyR2 that are involved in the intersubunit disulfide cross-linking and the channel's functional response to oxidative stress.

Cryo-EM snapshots and protein spin labeling in structural studies of HIV-1 Vpu.

We study the structure-function relationship of the HIV-1 encoded Vpu protein. HIV-1 causes HIV/AIDS leading to severe health complications. Understanding the molecular mechanisms of proteins that are key to the virus is important for the design of efficient therapeutics. Vpu forms ion-conducting oligomers in cellular membranes leading to cellular ion disbalance; its monomer is deemed functionally important as well, mostly through Vpu-host proteins interaction. However, the detailed structure of full-length (FL) Vpu in its oligomeric and monomeric states are currently poorly understood. Here, we describe our efforts to identify conditions under which we obtained high-quality cryo-EM data for structural determinations. In our studies, we used a chimera protein of maltose binding protein fused to the N-terminal of FL Vpu (MBP-Vpu). In initial assessments by negative staining EM, we found that MBP-Vpu forms stable oligomers, likely pentamers, in solution. Then, we subjected these pentamers to cryo-EM imaging and obtained high-quality data, which will be used in further structure reconstruction. We will also present cryo-EM data for MBP-Vpu oligomer residing in short-chain lipids, which will be useful to solve the structure of this protein in a lipid environment. Further, we will present results for the expression and purification of the Vpu C-terminal domain as a fusion construct with SUMO protein, and several single cysteine mutants located in the Vpu C-terminal helix 2, a protein region that associates with the membrane surface. These mutants will be spin-labeled and used in cw EPR studies to assess the conformational dynamics of FL Vpu and identify the membrane-contact sites in the Vpu C-terminal region. Overall, we made substantial progress towards characterizing the structure of FL Vpu.

Adagrasib: First Approval.

Adagrasib (KRAZATI™) is an orally available, potent, irreversible, small molecule inhibitor of KRAS G12C mutant isoform being developed by Mirati Therapeutics for the treatment of solid tumours harbouring KRAS G12C oncogenic driver mutation, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Adagrasib covalently binds to the mutant cysteine in KRAS G12C and locks the mutant KRAS protein in its inactive state, thereby preventing downstream signalling without affecting wild-type KRAS protein. In December 2022, adagrasib received its first approval in the USA for the treatment of adults with KRAS G12C-mutated locally advanced or metastatic NSCLC (as determined by an FDA approved test) who have received ≥1 prior systemic therapy. It was approved under accelerated approval based on objective response rate and duration of response, and its continued approval for this indication may be contingent upon verification and description of a clinical benefit in a confirmatory trial(s). The drug is under regulatory review for NSCLC in the European Union and is in development for CRC in the US. Clinical studies of adagrasib in solid tumours, including CRC, are underway in several countries. This article summarizes the milestones in the development of adagrasib leading to this first approval for KRAS G12C-mutated locally advanced or metastatic NSCLC.

Characterization of Mitochondrial Heat Shock Protein 60 variants in HEK293 Cells Transformed into Steroidogenic

Introduction: During pregnancy, P4 is essential to maintain the maternal-fetal relationship. Maternal cholesterol is the main source of P4 production, a process that takes place in the syncytiotrophoblast mitochondrion. The mechanism and proteins involved in the cholesterol transport for the steroidogenic process are still unknown in detail. The STARD3 protein could be the substitute for its STARD1 equivalent localized in all acute response tissues. However, mutation or null STARD3 mice maintain their reproductive capacity, suggesting other proteins are involved in this process. Previously, we reported that the HSP60 participates in steroidogenesis in mitochondria isolated from the placental syncytiotrophoblast, mitochondrial contact sites or JEG-3. Also, take relevance that non-steroidogenic cells, such as the HEK293, which are human kidney embryo cells, when are transformed into steroidogenic by transfection of the steroidogenic machinery, they synthesize progesterone. To understand better the mechanism through which HSP60 participates in placental steroidogenesis, mutation of cysteine 442, which is essential in the active site for its activity, and deletion of 146 amino acid residues of the N-terminal of HSP60 were performed. The first was implemented to determine whether the protein structure is essential to support steroidogenesis, and the second was done to elucidate whether its activity occurs outside or inside the mitochondrion. Methods: Two mutants were obtained: a) cysteine 442 was replaced by alanine (HSP60C442A) and b) the HSP60-mature (HSP60M) without the mitochondrial-leading sequence. Human kidney cells HEK293 were transformed into steroidogenic by transfection with pECE-P450scc, pCMV-3βHSD-I. The transfected cells were transfected with the HSP60wt, HSP60C442A, or HSP60M plasmids. The transfection was validated by western blot and P4 was determined by an enzyme immunoassay kit. HSP60 without mutations was used as control (HSP60wt). Results: The synthesis of P4 was stimulated by the wild type HSP60 (HSP60wt). However, with both mutants, steroidogenesis occurred as in the control, suggesting that mutants do not support P4 synthesis. Discussion: The mechanism to transport cholesterol to steroidogenic mitochondria requires the full HSP60 to support P4 synthesis, which is necessary to maintain pregnancy. Highlights HSP60 participates in the steroidogenesis of transformed HEK293 cells. Cys442 mutant of HSP60 loses its activity in steroidogenesis. N-terminal deletion of HSP60 is not involved in steroidogenesis. Native HSP60 is critical for steroidogenesis.