Palmitoylation at a conserved cysteine residue facilitates gasdermin D-mediated pyroptosis and cytokine release.

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1ß secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis.

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that exclusively labels isoAsp with deuterium. This method capitalizes on the unique structural characteristics of isoAsp residues, which possess a free α-carboxyl group and can form an oxazolone ring. Once the oxazolone ring forms, it facilitates racemization at the Cα-position, incorporating a deuteron from a D2O solvent. The sites of deuterium-incorporated isoAsp in proteins can be unequivocally determined by comparing the precursor and product ion masses of the peptides from proteins reacted in H2O and D2O. The effectiveness of this method has been demonstrated through its application to model proteins lysozyme and rituximab. Furthermore, we have confirmed that the isoAsp deuterium-labeling reaction efficiently labels both l- and d-isoAsp without distinction, as well as isoglutamic acid (isoGlu), for which no effective detection methods currently exist.

Significance of Histidine Hydrogen-Deuterium Exchange Mass Spectrometry in Protein Structural Biology.

Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determining the acid-base dissociation constant (Ka) of histidine imidazole rings in proteins often yields valuable insights into protein functions. Significant efforts have been dedicated to measuring the pKa values of histidine residues in various proteins, with nuclear magnetic resonance (NMR) spectroscopy being the most commonly used technique. However, NMR-based methods encounter challenges in assigning signals to individual imidazole rings and require a substantial amount of proteins. To address these issues associated with NMR-based approaches, a mass-spectrometry-based method known as histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) has been developed. This technique not only determines the pKa values of histidine imidazole groups but also quantifies their solvent accessibility. His-HDX-MS has proven effective across diverse proteins, showcasing its utility. This review aims to clarify the fundamental principles of His-HDX-MS, detail the experimental workflow, explain data analysis procedures and provide guidance for interpreting the obtained results.

High-Resolution Structural Proteomics of Mitochondria Using the 'Build and Retrieve' Methodology.

The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.

Vactosertib, a novel TGF-ß1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma.

Functional blockade of the transforming growth factor-beta (TGF-ß) signaling pathway improves the efficacy of cytotoxic and immunotherapies. We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF-ß type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma patients who had received ≥2 lines of chemoimmunotherapy. Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, while pomalidomide alone historically achieved 20% PFS-6. Following treatment, the immunosuppressive marker PD-1 expression was reduced on patient CD8+ T-cells. Following ex vivo treatment, vactosertib decreased PD-1 expression on patient CD138+ cells, reduced PD-L1/PD-L2 on patient CD138+ cells and enhanced the anti-myeloma activity of autologous T-cells. Taken together, vactosertib is a safe immunotherapy that modulates the T-cell immunophenotype to reinvigorate T-cell fitness. Multiple myeloma (MM) is a genetically heterogeneous hematologic malignancy characterized by the excessive proliferation of clonal plasma cells (1, 2). MM remains mostly incurable but a small group of patients can achieve long-term remission (3). Treatment of MM presents unique challenges due to the complex molecular pathophysiology and genetic heterogeneity (4, 5). Given that MM is the second most common blood cancer characterized by cycles of remission and relapse, the development of new therapeutic modalities is crucial (6, 7). The prognosis for MM patients has improved substantially over the past two decades with the development of more effective therapeutics, e.g., proteasome inhibitors, and regimens that demonstrate greater anti-tumor activity (8-10). The management of RRMM represents a vital aspect of the overall care for patients with disease and a critical area of ongoing scientific and clinical research (10-12).

High-resolution structural-omics of human liver enzymes.

We applied raw human liver microsome lysate to a holey carbon grid and used cryo-electron microscopy (cryo-EM) to define its composition. From this sample we identified and simultaneously determined high-resolution structural information for ten unique human liver enzymes involved in diverse cellular processes. Notably, we determined the structure of the endoplasmic bifunctional protein H6PD, where the N- and C-terminal domains independently possess glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase enzymatic activity, respectively. We also obtained the structure of heterodimeric human GANAB, an ER glycoprotein quality-control machinery that contains a catalytic α subunit and a noncatalytic ß subunit. In addition, we observed a decameric peroxidase, PRDX4, which directly contacts a disulfide isomerase-related protein, ERp46. Structural data suggest that several glycosylations, bound endogenous compounds, and ions associate with these human liver enzymes. These results highlight the importance of cryo-EM in facilitating the elucidation of human organ proteomics at the atomic level.

MYO10 regulates genome stability and cancer inflammation through mediating mitosis.

Genomic instability can promote inflammation and tumor development. Previous research revealed an unexpected layer of regulation of genomic instability by a cytoplasmic protein MYO10; however, the underlying mechanism remained unclear. Here, we report a protein stability-mediated mitotic regulation of MYO10 in controlling genome stability. We characterized a degron motif and phosphorylation residues in the degron that mediate ß-TrCP1-dependent MYO10 degradation. The level of phosphorylated MYO10 protein transiently increases during mitosis, which is accompanied by a spatiotemporal cellular localization change first accumulating at the centrosome then at the midbody. Depletion of MYO10 or expression of MYO10 degron mutants, including those found in cancer patients, disrupts mitosis, increases genomic instability and inflammation, and promotes tumor growth; however, they also increase the sensitivity of cancer cells to Taxol. Our studies demonstrate a critical role of MYO10 in mitosis progression, through which it regulates genome stability, cancer growth, and cellular response to mitotic toxins.

Simultaneous solving high-resolution structures of various enzymes from human kidney microsomes.

The ability to investigate tissues and organs through an integrated systems biology approach has been thought to be unobtainable in the field of structural biology, where the techniques mainly focus on a particular biomacromolecule of interest. Here we report the use of cryo-electron microscopy (cryo-EM) to define the composition of a raw human kidney microsomal lysate. We simultaneously identify and solve cryo-EM structures of four distinct kidney enzymes whose functions have been linked to protein biosynthesis and quality control, biosynthesis of retinoic acid, gluconeogenesis and glycolysis, and the regulation and metabolism of amino acids. Interestingly, all four of these enzymes are directly linked to cellular processes that, when disrupted, can contribute to the onset and progression of diabetes. This work underscores the potential of cryo-EM to facilitate tissue and organ proteomics at the atomic level.

Toward structural-omics of the bovine retinal pigment epithelium.

The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the composition of a raw bovine retinal pigment epithelium (RPE) lysate. From this sample, we simultaneously identify and solve cryo-EM structures of seven different RPE enzymes whose functions affect neurotransmitter recycling, iron metabolism, gluconeogenesis, glycolysis, axonal development, and energy homeostasis. Interestingly, dysfunction of these important proteins has been directly linked to several neurodegenerative disorders, including Huntington's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and schizophrenia. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

53BP1 regulates heterochromatin through liquid phase separation.

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin protein HP1α in a mutually dependent manner. Deletion of 53BP1 results in a reduction in heterochromatin centers and the de-repression of heterochromatic tandem repetitive DNA. We identify domains and residues of 53BP1 required for its LLPS, which overlap with, but are distinct from, those involved in DSB repair. Further, 53BP1 mutants deficient in DSB repair, but proficient in LLPS, rescue heterochromatin de-repression and protect cells from stress-induced DNA damage and senescence. Our study suggests that in addition to DSB repair modulation, 53BP1 contributes to the maintenance of heterochromatin integrity and genome stability through LLPS.

Identifying Protein-Drug Interactions in Cell Lysates Using Histidine Hydrogen Deuterium Exchange.

Identifying the targets of a drug is critical to understand the mechanism of action and predicts possible side effects. The conventional approach is capturing interacting proteins by affinity purification. However, it requires drugs to be immobilized to a solid support or derivatized with chemical moieties used for pulling down interacting proteins. Such covalent modifications to drugs may mask a critical recognition site for or alter the binding affinity to their targets. To overcome the drawback, several methods that do not require covalent modifications to drugs have been developed. These methods identify targets by detecting proteins whose thermodynamic stability is enhanced in the presence of drugs. Although the utility of these methods has been demonstrated, the difficulty in identifying low abundant targets is the common problem of these methods. We have developed a new target identification method that increases the likelihood of identifying low abundant targets. The method uses histidine-hydrogen deuterium exchange (His-HDX) as a readout technique to probe the changes in protein stability induced by drugs. The workflow involves incubating cell lysates in various concentrations of a protein denaturant in the presence and absence of a drug in D2O followed by digestion of the proteins, enrichment of His-containing peptides, and analysis of the enriched His-peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method was successfully applied to identify the interaction between endogenously expressed MAPK14 and its inhibitor in HEK293 cell lysates. The implementation of selective enrichment of histidine-containing peptides in the workflow was a key that enabled identifying the MAPK14-inhibitor interaction.

Translational regulation in the brain by TDP-43 phase separation.

The in vivo physiological function of liquid-liquid phase separation (LLPS) that governs non-membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain function. Brain neurons of these mice, however, did not show TDP-43 proteinopathy or neurodegeneration. Instead, the global rate of protein synthesis was found to be greatly enhanced by TDP-43 LLPS loss. Mechanistically, TDP-43 LLPS ablation increased its association with PABPC4, RPS6, RPL7, and other translational factors. The physical interactions between TDP-43 and translational factors relies on a motif, the deletion of which abolished the impact of LLPS-deficient TDP-43 on translation. Our findings show a specific physiological role for TDP-43 LLPS in the regulation of brain function and uncover an intriguing novel molecular mechanism of translational control by LLPS.

Photoreceptor Cell Calcium Dysregulation and Calpain Activation Promote Pathogenic Photoreceptor Oxidative Stress and Inflammation in Prodromal Diabetic Retinopathy.

This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy.

SLX4IP promotes RAP1 SUMOylation by PIAS1 to coordinate telomere maintenance through NF-κB and Notch signaling.

The maintenance of telomere length supports repetitive cell division and therefore plays a central role in cancer development and progression. Telomeres are extended by either the enzyme telomerase or the alternative lengthening of telomeres (ALT) pathway. Here, we found that the telomere-associated protein SLX4IP dictates telomere proteome composition by recruiting and activating the E3 SUMO ligase PIAS1 to the SLX4 complex. PIAS1 SUMOylated the telomere-binding protein RAP1, which disrupted its interaction with the telomere-binding protein TRF2 and facilitated its nucleocytoplasmic shuttling. In the cytosol, RAP1 bound to IκB kinase (IKK), resulting in activation of the transcription factor NF-κB and its induction of Jagged-1 expression, which promoted Notch signaling and the institution of ALT. This axis could be targeted therapeutically in ALT-driven cancers and in tumor cells that develop resistance to antitelomerase therapies. Our results illuminate the mechanisms underlying SLX4IP-dependent telomere plasticity and demonstrate the role of telomere proteins in directly coordinating intracellular signaling and telomere maintenance dynamics.

Site-Specific Labeling and 19F NMR Provide Direct Evidence for Dynamic Behavior of the Anthrax Toxin Pore ϕ-Clamp Structure.

The anthrax toxin protective antigen (PA), the membrane binding and pore-forming component of the anthrax toxin, was studied using 19F NMR. We site-specifically labeled PA with p-fluorophenylalanine (pF-Phe) at Phe427, a critically important residue that comprises the ϕ-clamp that is required for translocation of edema factor (EF) and lethal factor (LF) into the host cell cytosol. We utilized 19F NMR to follow low-pH-induced structural changes in the prepore, alone and bound to the N-terminal PA binding domain of LF, LFN. Our studies indicate that pF-Phe427 is dynamic in the prepore state and then becomes more dynamic in the transition to the pore. An increase in dynamic behavior at the ϕ-clamp may provide the necessary room for movement needed in translocating EF and LF into the cell cytosol.

Indole-3-acetic acid synthesized through the indole-3-pyruvate pathway promotes Candida tropicalis biofilm formation.

We previously found that the elevated abundance of the fungus Candida tropicalis is positively correlated with the bacteria Escherichia coli and Serratia marcescens in Crohn's disease patients and the three pathogens, when co-cultured, form a robust mixed-species biofilm. The finding suggests that these three pathogens communicate and promote biofilm formation, possibly through secretion of small signaling molecules. To identify candidate signaling molecules, we carried out a metabolomic analysis of the single-species and triple-species cultures of the three pathogens. This analysis identified 15 metabolites that were highly increased in the triple-species culture. One highly induced metabolite was indole-3-acetic acid (IAA), which has been shown to induce filamentation of certain fungi. We thus tested the effect of IAA on biofilm formation of C. tropicalis and demonstrated that IAA promotes biofilm formation of C. tropicalis. Then, we carried out isotope tracing experiments using 13C-labeled-tryptophan as a precursor to uncover the biosynthesis pathway of IAA in C. tropicalis. The results indicated that C. tropicalis synthesizes IAA through the indole-3-pyruvate pathway. Further studies using inhibitors of the indole-3-pyruvate pathway are warranted to decipher the mechanisms by which IAA influences biofilm formation.

The promyelocytic leukemia protein isoform PML1 is an oncoprotein and a direct target of the antioxidant sulforaphane (SFN).

The gene encoding promyelocytic leukemia protein (PML) generates several spliced isoforms. Ectopic expression of PML1 promotes the proliferation of ERα-positive MCF-7 breast cancer (BC) cells, while a loss of PML by knockdown or overexpression of PML4 does the opposite. PML is an essential constituent of highly dynamic particles called PML nuclear bodies (NBs). PML NBs are heterogenous multiprotein subnuclear structures that are part of cellular stress sensing machinery. The antioxidant sulforaphane (SFN) inhibits the proliferation of BC cells and causes a redistribution of the subcellular localization of PML, a disruption of disulfide-bond linkages in nuclear PML-containing complexes, and a reduction in the number and size of PML NBs. Mechanistically, SFN modifies several cysteine residues, including C204, located in the RBCC domain of PML. PML is sumoylated and contains a Sumo-interacting motif, and a significant fraction of Sumo1 and Sumo2/3 co-localizes with PML NBs. Ectopic expression of the mutant C204A selectively inhibits the biogenesis of endogenous PML NBs but not PML-less Sumo1-, Sumo2/3, or Daxx-containing nuclear speckles. Importantly, PML1 (C204A) functions as a dominant-negative mutant over endogenous PML protein and promotes anti-proliferation activity. Together, we conclude that SFN elicits its cytotoxic activity in part by inactivating PML1's pro-tumorigenic activity.