Single Cysteine Research Articles

Cellular Uptake of Tau Aggregates Triggers Disulfide Bond Formation in Four-Repeat Tau Monomers.

Oxidative stress is an important driver of aging and has been linked to numerous neurodegenerative disorders, including Alzheimer's disease. A key pathological hallmark of Alzheimer's are filamentous inclusions made of the microtubule associated protein Tau. Based on alternative splicing, Tau protein can feature either three or four microtubule binding repeats. Distinctively, three-repeat Tau contains a single cysteine; four-repeat Tau contains two. Although there is evidence that the cysteines in pathological Tau filaments exist in the reduced form, very little is known about the alternative disulfide-bonded state. It is unclear whether it can exist nontransiently in the reducing environment of the cytosol. Such knowledge, however, is important as different redox states of Tau could modulate aggregation. To address this question, we transfected HEK293 cells expressing the P301S variant of four-repeat Tau with fibril seeds composed of compact, disulfide-bonded Tau monomers. In vitro, these fibrils are observed to recruit only compact Tau, but not Tau in which the cysteines are reduced or replaced by alanines or serines. In line with this characteristic, the fibrils dissociate when treated with a reducing agent. When offered to HEK293 cells, variant Tau protein is recruited to the seeds forming intracellular fibrils with the same seeding properties as the in vitro counterparts. Markedly, the proteins in these fibrils have a compact, disulfide-bonded configuration and dissociate upon reduction. These findings reveal that uptake of exogeneous fibril seeds triggers oxidation of Tau monomers, modulating intracellular aggregation.

Multi-Site Red-Edge Excitation Shift Reveals the Residue-Specific Solvation Dynamics during the Native to Amyloid-like Transition of an Amyloidogenic Protein.

Changes in water-protein interactions are crucial for proteins to achieve functional and nonfunctional conformations during structural transitions by modulating local stability. Amyloid-like protein aggregates in deteriorating neurons are hallmarks of neurodegenerative disorders. These aggregates form through significant structural changes, transitioning from functional native conformations to supramolecular cross-β-sheet structures via misfolded and oligomeric intermediates in a multistep process. However, the site-specific dynamics of water molecules from the native to misfolded conformations and further to oligomeric and compact amyloid structures remain poorly understood. In this study, we used the fluorescence method known as red-edge excitation shift (REES) to investigate the solvation dynamics at specific sites in various equilibrium conformations en route to the misfolding and aggregation of the functional domain of the TDP-43 protein (TDP-43tRRM). We generated three single tryptophan-single cysteine mutants of TDP-43tRRM, with the cysteines at different positions and tryptophan at a fixed position. Each sole cysteine was fluorescently labeled and used as a site-specific fluorophore along with the single tryptophan, creating four monitorable sites for REES studies. By investigating the site-specific extent of REES, we developed a residue-specific solvation dynamics map of TDP-43tRRM during its misfolding and aggregation. Our observations revealed that solvation dynamics progressively became more rigid and heterogeneous to varying extents at different sites during the transition from native to amyloid-like conformations.

Thiol-Based Redox Molecules: Potential Antidotes for Acrylamide Toxicity.

Acrylamide (ACR) is a low-molecular weight, non-aromatic reagent, widely used in industry, such as in the manufacture of paper, textiles, plastics, cosmetics, and dyes. ACR is formed during the cooking of starchy food and its toxicity results mainly by conferring oxidative stress by elevating reactive oxygen species (ROS). To identify potential antidotes for ACR toxicity, we evaluated the efficacy of several thiol-based molecules known for ROS-scavenging, disulfide-reducing properties, and inhibition of oxidative stress-induced activation of the mitogen-activated protein kinases (MAPKs): the extracellular-signal-regulated-kinases (ERK1/2), p38-mitogen-activated-protein-kinases (p38MAPK), and c-Jun-N-terminal-kinases (JNKs). We established a reproducible assay testing N-acetylcysteine (NAC), AD4/NACA, and the N-and C-blocked tri- and tetra-thioredoxin-mimetic (TXM) peptides, in PC12 cells. Our results demonstrate that these compounds exhibited high efficacy in suppressing ACR-induced MAPK activation, either prior to or subsequent to ACR exposure. The inhibition by single cysteine (Cys) residue, NAC and AD4/NACA (NAC-amide), 2 Cys peptides TXM-CB30, AcDCys-Gly-DCysNH2, TXM-CB20, AcCys-Gly-CysNH2, SuperDopa (SD, Ac-CysL-Levodopa-CysNH2, TXM-CB13, AcCys-Met-Lys-CysNH2, and a 3-Cys peptide, TXM-CB16, AcCys-γGlu-Cys-CysNH2 was dose-dependent and potency displayed a direct correlation with the number of Cys residues. Cellular proteolysis of SD, which consists of levodopa flanked by two Cys, may suppress the manifestation of Parkinson's disease (PD)-like symptoms mediated by chronic ACR exposure not only through lowering oxidative stress but also by replenishing cellular levels of dopamine. Overall, these results could advance the clinical application of TXM peptides as potential treatments for acute and/or chronic exposure to ACR and show promise as antidotes for preventing ACR-triggered PD-like neurotoxic symptoms.

Blocking GP130 binding in interleukin-11 through site-specific PEGylation attenuates bleomycin-induced pulmonary fibrosis in mice

Recent insights have identified interleukin-11 (IL-11) as a pivotal profibrotic cytokine, with its signaling through IL-11Rα and GP130 receptors emerging as a promising therapeutic target for fibrotic diseases. Herein, we developed receptor-biased IL-11 via site-specific PEGylation at the GP130 binding interface, aiming to explore its therapeutic potential for bleomycin-induced pulmonary fibrosis in mice. By conducting single site-directed cysteine mutagenesis at site II or site III of IL-11, we refined the conjugation site, demonstrating that mutation at site III exhibits heightened sensitivity to GP130 binding and signaling. Cysteine-based PEGylation substantially attenuated the ability of GP130 to bind to IL-11 W147C, while almost entirely preserving its IL-11Rα binding ability. These PEGylated IL-11 W147C analogs showed potent inhibition of TF-1 cell proliferation and significant antagonism to TGF-β1-induced human lung fibroblasts (HLFs) differentiation into myofibroblasts. Moreover, PEGylation significantly prolonged the half-life of IL-11 W147C in healthy rats. Subcutaneous administration of PEGylated analogs, particularly PEG20/40 k-IL-11 W147C, effectively mitigated extracellular matrix deposition, preserved alveolar architecture, and attenuated the progression of pulmonary fibrosis in mice. The finding of this study not only underscores the therapeutic potential of IL-11 modulation, but also provides a general strategy for the design of cytokine-based biased antagonists and agonists targeting these multifaceted signaling pathways.

Cohesive Living Bacterial Films with Tunable Mechanical Properties from Cell Surface Protein Display.

Engineered living materials (ELMs) constitute a novel class of functional materials that contain living organisms. The mechanical properties of many such systems are dominated by the polymeric matrices used to encapsulate the cellular components of the material, making it hard to tune the mechanical behavior through genetic manipulation. To address this issue, we have developed living materials in which mechanical properties are controlled by the cell-surface display of engineered proteins. Here, we show that engineered Esherichia coli cells outfitted with surface-displayed elastin-like proteins (ELPs, designated E6) grow into soft, cohesive bacterial films with biaxial moduli around 14 kPa. When subjected to bulge-testing, such films yielded at strains of approximately 10%. Introduction of a single cysteine residue near the exposed N-terminus of the ELP (to afford a protein designated CE6) increases the film modulus 3-fold to 44 kPa and eliminates the yielding behavior. When subjected to oscillatory stress, films prepared from E. coli strains bearing CE6 exhibit modest hysteresis and full strain recovery; in E6 films much more significant hysteresis and substantial plastic deformation are observed. CE6 films heal autonomously after damage, with the biaxial modulus fully restored after a few hours. This work establishes an approach to living materials with genetically programmable mechanical properties and a capacity for self-healing. Such materials may find application in biomanufacturing, biosensing, and bioremediation.

Site-specific dimerization of interleukin-11 alleviates bleomycin-induced pulmonary fibrosis in mice

Interleukin-11 (IL-11) has recently been identified as a critical profibrotic cytokine, and IL-11 signaling pathway via IL-11Rα and GP130 receptors has been shown to be a promising therapeutic target for the treatment of fibrotic diseases. Herein, we devised two kinds of IL-11 dimer with receptor-biased binding ability through site-specific crosslinking at the interface involving GP130 binding and signaling, aiming to explore their therapeutic potentials for bleomycin-induced pulmonary fibrosis in mice. A single cysteine mutation at site W147 of human IL-11 (IL-11 W147C) was conducted for site-specific crosslinking. The ability of GP130 to bind to IL-11 W147C dimer was substantially weakened by cysteine-based dimerization, while the ability of IL-11 W147C dimer to bind to IL-11Rα was almost entirely preserved or even enhanced. The IL-11 W147C dimer potently inhibited TF-1 cell proliferation and TGF-β1-induced human lung fibroblast differentiation into myofibroblasts. We also showed that dimerization substantially extended the circulation time of IL-11 W147C dimer in healthy rats. Subcutaneous administration of IL-11 W147C dimer significantly reduced extracellular matrix deposition, preserved alveolar architecture and alleviated pulmonary fibrosis development in mice. The findings of this study may provide a general strategy for the design of cytokine-based receptor-biased antagonists and agonists targeting these multifaceted signaling pathways.

Native Mass Spectrometry Reveals Binding Interactions of SARS-CoV-2 PLpro with Inhibitors and Cellular Targets.

Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function. A PLpro mutant lacking a single cysteine was used to localize inhibitor binding, and thermodynamic measurements revealed that inhibitor PR-619 stabilized the folded PLpro structure. These results will inform further development of PLpro as a therapeutic target against SARS-CoV-2 and other emerging coronaviruses.

Chemically Tagging Cargo for Specific Packaging inside and on the Surface of Virus-like Particles.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism

Basic Helix-Loop-Helix (bHLH) transcription factors TFEB/TFE3 and HLH-30 are key regulators of autophagy induction and lysosomal biogenesis in mammals and C. elegans, respectively. While much is known about the regulation of TFEB/TFE3, how HLH-30 subcellular dynamics and transactivation are modulated are yet poorly understood. Thus, elucidating the regulation of C. elegans HLH-30 will provide evolutionary insight into the mechanisms governing the function of bHLH transcription factor family. We report here that HLH-30 is retained in the cytoplasm mainly through its conserved Ser201 residue and that HLH-30 physically interacts with the 14-3-3 protein FTT-2 in this location. The FoxO transcription factor DAF-16 is not required for HLH-30 nuclear translocation upon stress, despite that both proteins partner to form a complex that coordinately regulates several organismal responses. Similar as described for DAF-16, the importin IMB-2 assists HLH-30 nuclear translocation, but constitutive HLH-30 nuclear localization is not sufficient to trigger its distinctive transcriptional response. Furthermore, we identify FTT-2 as the target of diethyl maleate (DEM), a GSH depletor that causes a transient nuclear translocation of HLH-30. Together, our work demonstrates that the regulation of TFEB/TFE3 and HLH-30 family members is evolutionarily conserved and that, in addition to a direct redox regulation through its conserved single cysteine residue, HLH-30 can also be indirectly regulated by a redox-dependent mechanism, probably through FTT-2 oxidation.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

Insights into the production and evolution of lantibiotics from a computational analysis of peptides associated with the lanthipeptide cyclase domain.

Lanthipeptides are a large group of ribosomally encoded peptides cyclized by thioether and methylene bridges, which include the lantibiotics, lanthipeptides with antimicrobial activity. There are over 100 experimentally characterized lanthipeptides, with at least 25 distinct cyclization bridging patterns. We set out to understand the evolutionary dynamics and diversity of lanthipeptides. We identified 977 peptides in 2785 bacterial genomes from short open-reading frames encoding lanthipeptide modifiable amino acids (C, S and T) that lay chromosomally adjacent to genes encoding proteins containing the cyclase domain. These appeared to be synthesized by both known and novel enzymatic combinations. Our predictor of bridging topology suggested 36 novel-predicted topologies, including a single-cysteine topology seen in 179 lanthionine or labionin containing peptides, which were enriched for histidine. Evidence that supported the relevance of the single-cysteine containing lanthipeptide precursors included the presence of the labionin motif among single cysteine peptides that clustered with labionin-associated synthetase domains, and the leader features of experimentally defined lanthipeptides that were shared with single cysteine predictions. Evolutionary rate variation among peptide subfamilies suggests that selection pressures for functional change differ among subfamilies. Lanthipeptides that have recently evolved specific novel features may represent a richer source of potential novel antimicrobials, since their target species may have had less time to evolve resistance.

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during βAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells.

The ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Cysteine oxidation as a regulatory mechanism of Arabidopsis plastidial NAD-dependent malate dehydrogenase.

Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)-dependent-oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD-dependent MDH (plNAD-MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD-MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD-MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD-MDH. In addition, a conformational change upon oxidation could be observed via non-reducing SDS-PAGE. Both effects, its inhibition and conformational change, were reversible by re-reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD-MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD-MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD-MDH activity or structure. Our results demonstrate that plNAD-MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co-regulated by the availability of its cofactors in vivo.

Abstract 731: pHLIP targeted intracellular delivery of calicheamicin

Abstract Calicheamicin is a potent, cell-cycle independent enediyne antibiotic that binds and cleaves DNA. Toxicity has led to its approved use in a targeted form as antibody-drug conjugates (ADC) for the treatment of liquid tumors. Mylotarg, one of the first such ADCs, was voluntarily withdrawn from the market due to safety concerns. However, Mylotarg was recently reapproved for acute myeloid leukemia based on new data that demonstrated efficacy with an acceptable safety profile. Besponsa, a related ADC, was approved for acute lymphocytic leukemia in 2017. In both agents, Mylotag and Besponsa, calicheamicin is conjugated to antibodies via an acid-labile hydrazone linker. We used a reduced calicheamicin to conjugate it to a single cysteine residue at the membrane-inserting end of a pH Low Insertion Peptide (pHLIP) via a disulfide bond to generate a linkerless cytoplasmic release. The cytoplasmic reduction of the disulfide releases the calicheamicin, which leads to its activation, DNA binding, and strand scission. pHLIP is a tumor-targeting agent now in clinical trials with imaging and therapeutic payloads. We studied the interaction of pHLIP-calicheamicin with liposomal and cellular membranes and demonstrated that the agent exhibits cytotoxic activity both in highly proliferative cancer cells and in non-proliferative immune cells, such as polarized M2 macrophages. In mice, treatment led to the growth inhibition of immuno-suppressive CT26 tumors with no signs of toxicity. Biodistribution studies confirmed tumor targeting with no accumulation of the agent in organs and tissues. The fluorescent version of the agent was observed within the tumor mass and tumor-stroma interface, where CD206+ M2 tumor-associated macrophages (M2-TAMs) reside. Treatment of tumors led to the depletion of M2-TAMs within the tumor core. Thus, pHLIP-calicheamicin could be developed into an effective therapeutic for the treatment of solid tumors with abundant immuno-suppressive M2-TAMs. Citation Format: Michael DuPont, Craig Klumpp, Marissa Iraca, Dana Allababidi, Hannah Visca, Donald Engelman, Oleg Andreev, Anna Moshnikova, Yana Reshetnyak. pHLIP targeted intracellular delivery of calicheamicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 731.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production

Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved single cysteine compound N-acetylcysteine (NAC) reduced GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Normal brain cells and other cancer cells showed no response to NAC. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. From analysis of the clinical proteomic tumor analysis consortium (CPTAC) database, we found that GBM cells exhibit lower expression of mitochondrial redox enzymes than four other cancers whose proteomic data are available in CPTAC. Knockdown of mitochondrial thioredoxin-2 in lung cancer cells induced NAC susceptibility, indicating the importance of mitochondrial redox enzyme expression in mitigating reductive stress. Intraperitoneal treatment of mice bearing orthotopic GBM xenografts with a two-cysteine peptide induced H2O2 in brain tumors in vivo. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

Acylation of MLKL Impacts Its Function in Necroptosis.

Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization, which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize the acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, with C184, C269, and C286 as possible acylation sites. Using all-atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane levels. Through investigations of the S-palmitoyltransferases that might acylate pMLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating the necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.

Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151. Arabinoxylan titration experiments with single cysteine mutants N54C and N151C labeled with the thiol-reactive fluorophore acrylodan or the ESR spin-label MTSSL validated the MDS results. Ligand binding at the SBS around Asn54 confirms previous reports, and analysis of the second SBS around N151C discovered in the present study includes residues Val98/Ala192/Ser155/His156. Understanding the regulation of xylanases contributes to efforts for industrial decarbonization and to establishing a sustainable energy matrix.