RAS proteins are critical signaling nodes that regulate cellular growth, with activating mutations in KRAS found in approximately 25% of human cancers. These mutations lead to chronic KRAS activation and sustained oncogenic signaling. Among >130 cancer-associated point mutations in KRAS, 99% cluster at hotspots (G12, G13, and Q61), with G12C mutations predominant in non-small cell lung cancer (NSCLC, ∼80%). The G12C mutation introduces a reactive cysteine, enabling covalent drug discovery strategies. Therapeutics such as AMG510 (Sotorasib) target KRASG12C by locking it in an inactive state; however, resistance arises in ∼60% of treated patients. To address this challenge, we aim to investigate how oxidation of KRAS and at Cys12 inKRASG12C influences its ability to bind covalent inhibitors, potentially offering insights into resistance mechanisms and novel therapeutic strategies. To explore the effects of oxidative modifications on KRASG12C function, we have started by engineering a recombinant KRASG12C protein that lacks other cysteines (denoted KRASG12C-CL) and introduced a Trp substitution (Y137W) to enable fluorescent kinetic analyses (KRASG12C-CLW; CL stands for cysteine-light). Recombinant protein samples were exposed to oxidants (H2O2) in the presence or absence of GDP, subsequently desalted using Bio-Spin Chromatography columns, and then analyzed by mass spectrometry to quantify the kinetics of oxidation to sulfinic acid state (-SO2H) under these conditions. Further fluorescent kinetic assays of reduced and oxidized KRASG12C with AMG510 and other KRAS inhibitors showed complex profiles distinguishing inhibitor binding to the reduced and oxidized KRASG12C. Additionally, we conducted targeted analyses of endogenous KRASG12C oxidation in Calu-1 non-small cell lung cancer cells. Calu-1 cells treated with 1 mM H2O2for 5 minutes demonstrated rapid oxidation of KRASG12C. Data will be presented showing the impact of KRASG12C oxidation on the therapeutic efficacy of clinical and experimental inhibitors. These approaches aim to better evaluate the impact of KRASG12C oxidation state on inhibitor engagement and therapeutic outcomes, addressing mechanisms of resistance and optimizing treatment strategies. Yismeilin R. Feliz Mosquea, Maria Luiza Carneiro Buchele, Derek Parsonage, Jingyun Lee, Surbhi Mundra, Sharon L. Campbell, Leslie B. Poole, Cristina M. Furdui. Impact of KRASG12C oxidation state on non-small-cell lung cancer response to targeted therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6997.

Subunit or inactivated vaccines comprise the majority of vaccines used against viral and bacterial pathogens. However, compared to their live/attenuated counterparts, these vaccines often demonstrate reduced immunogenicity, requiring multiple boosters and or adjuvants to elicit protective immune responses. For this reason, studies of adjuvants and the mechanism through which they can improve inactivated vaccine responses are critical for the development of vaccines with increased efficacy. Studies have shown that the direct conjugation of adjuvant to antigen promotes vaccine immunogenicity, with the advantage of both the adjuvant and antigen targeting the same cell. Using this strategy of direct linkage, we developed an inactivated influenza A (IAV) vaccine that is directly conjugated with the Toll-like receptor 7/8 agonist resiquimod (R848) through a heterobifunctional crosslinker. Previously, we showed that this vaccine resulted in improved protection and viral clearance in newborn nonhuman primates compared to a non-adjuvanted vaccine. We subsequently discovered that the choice of linker used to conjugate R848 to the virus alters the stimulatory activity of the vaccine, promoting increased maturation and proinflammatory cytokine production from DC differentiated in vitro. With this knowledge, we explored how the choice of crosslinker impacts the stimulatory activity of these vaccines. We found that the linker choice alters signaling through the NF-κB pathway in human monocyte-derived dendritic cells (moDCs). Further, we extended our analyses to in vivo differentiated APC present in human peripheral blood, replicating the linker-dependent differences found in in vitro differentiated cells. Finally, we demonstrated in a mouse model that the choice of linker impacts the amount of IAV-specific IgG antibody produced in response to vaccination. These data enhance our understanding of conjugation approaches for improving vaccine immunogenicity.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are dysregulated in many pervasive diseases. Recently, we discovered that ERK1/2 is oxidized by signal-generated hydrogen peroxide in various cell types. Since the putative sites of oxidation lie within or near ERK1/2's ligand-binding surfaces, we investigated how oxidation of ERK2 regulates interactions with the model substrates Sub-D and Sub-F. These studies revealed that ERK2 undergoes sulfenylation at C159 on its D-recruitment site surface and that this modification modulates ERK2 activity differentially between substrates. Integrated biochemical, computational, and mutational analyses suggest a plausible mechanism for peroxide-dependent changes in ERK2-substrate interactions. Interestingly, oxidation decreased ERK2's affinity for some D-site ligands while increasing its affinity for others. Finally, oxidation by signal-generated peroxide enhanced ERK1/2's ability to phosphorylate ribosomal S6 kinase A1 (RSK1) in HeLa cells. Together, these studies lay the foundation for examining crosstalk between redox- and phosphorylation-dependent signaling at the level of kinase-substrate selection.

ABSTRACTThe development of mutant-selective inhibitors for the KRASG12C allele has generated considerable excitement. These KRASG12C inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain, motivating the present studies. Measurement of the C12 thiol pKa by NMR and an independent biochemical assay found a depressed pKa (relative to free cysteine) of 7.6 consistent with its susceptibility to chemical ligation. Using a novel and validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that ARS-853 addition at 5°C elicited both a rapid first phase (attributed to binding, yielding a Kd of 36.0 ± 0.7 μM), and a second, slower pH-dependent phase taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C thiol to sulfinic acid alters RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

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) similar to 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 mu M) 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.

Additional file 3. Identification of glycoglycerolipids in lipid extracts of wild type and genetically-engineered cyanobacteria.

Background Dietary omega-3 (n-3), long chain (LC-, ≥ 20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18-carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems. Results We utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding Δ15 and Δ6 desaturases, respectively) and “vesicle-inducing protein in plastids” (Vipp1) to induce production of stearidonic acid (SDA, 18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and ~ 99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules. Conclusions Overall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (C-P, C-R). The first step in catalysis, the reduction of peroxide substrate, is fast, 10(7) - 10(8) M(-1)s(-1) for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of C-P, which occurs when a second molecule of peroxide reacts with the C-P in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the C-P sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Antibiotics with novel mechanisms of action are desperately needed to combat the increasing rates of multidrug-resistant infections. Bacterial pantothenate kinase (PanK) has emerged as a target of interest to cut off the biosynthesis of coenzyme A. Herein we report the results of an in vitro high-throughput screen of over 10 000 small molecules against Bacillus anthracis PanK, as well as a follow-up screen of hits against PanK isolated from Pseudomonas aeruginosa and Burkholderia cenocepacia. Nine hits are structurally categorized and analyzed to set the stage for future drug development.

2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-angstrom-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo. These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of sulfenic acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase.

AIMS:Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx. RESULTS:Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4-5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pKa values of the peroxidatic cysteine were also shifted up by 1-1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants-T77V, T43A, and T43S-exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants were solved and many showed alterations in stability of the fully folded active site loop. INNOVATION:This is the first study of Prx mutants to comprehensively assess the effects of mutations on catalytic activities, the active site cysteine pKa, and the protein structure and oligomeric status. CONCLUSION:The Arg119 side chain must be properly situated for efficient catalysis, but for other debilitating variants, the functional defects could be explained by structural perturbations and/or associated decamer destabilization rather than direct effects. This underscores the importance of our comprehensive approach. A remarkable new finding was the preference of the reductant for decamers. Antioxid. Redox Signal. 28, 521-536.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Extracellular regulated kinase 2 (ERK2), which is the terminal serine/threonine protein kinase in the Ras-Raf-MEK-ERK signaling axis, is involved in the regulation of many cellular processes, including cell proliferation, differentiation, survival, and cell cycle progression. Recently, ERK2 was shown to undergo reversible oxidation, inhibiting reactivity toward Elk1 substrate, following growth factor stimulation in a number of cell types (Keyes et al., FRBM, 2017). Interestingly, one of the residues exhibiting oxidation sensitivity (C159), is located within the D-recruitment site (DRS). Since the DRS is involved in binding a subset of ERK2 substrates, we compared the ability of wild-type ERK2 and ERK2(C159S) to phosphorylate a model DRS peptide substrate, Sub-D, following treatment with various concentrations of H2O2. Kinetic analysis demonstrated that, while both wild-type ERK2 and ERK2(C159S) exhibit similar apparent kcat and Km values for Sub-D in the absence of H2O2, treatment with H2O2 causes more significant changes in kcat,app and Km,app for Sub-D in wild-type compared with the C159S mutant of ERK2. Together, these data suggest that redox modification of ERK2 may alter its ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter ERK2's activity toward some, but not all, of its downstream substrates. To explore this possibility further, we investigated the impact of redox modification on ERK2's global substrate selectivity using functional protein microarrays. Consistent with our hypothesis, ERK2-mediated phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2-dependent changes in their phosphorylation status, indicating that C159 oxidation controls ERK activity by modulating substrate specificity. Together, these studies offer important insights into redox regulation of ERK2 and provide potential points of signal integration between kinase- and redox-dependent signaling pathways.