All organisms have thioredoxin reductase (TR) or glutathione reductase (GR), the only enzymes that use reduced nicotinamide adenine dinucleotide phosphate to reduce cytosolic disulfides into thiols, thereby powering deoxyribonucleotide biosynthesis, elimination of oxidants, oxidative damage repair and reduction of the disulfide nutrient cystine into the thiol amino acid cysteine. Hence, TR/GR-null bacteria or yeast are inviable; yet, remarkably, mice with TR/GR-null livers thrive, in part by synthesizing life-sustaining cysteine through alternative pathways that evolved in metazoans. Although TR/GR-null livers generate some of their cysteine through the serine transsulfuration pathway, we here show that most cysteine in TR/GR-null livers comes from a pathway in which pyridoxal-phosphate-dependent cleavage of a carbon-sulfur bond in cystine generates cysteine persulfide, which decomposes nonenzymatically into cysteine. This potent yet previously unrecognized pathway is regulated by cellular levels of sulfur metabolites and represents a potent cytoprotective response that might be induced in most mammalian cells under conditions that chronically elevate cytosolic cystine levels.

The recent emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions and the limited availability of established cell culture models. Primary bovine embryonic fibroblasts (BeEFs) were isolated and investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles, infectious virus production, viral replication, and plaque morphology in BeEFs following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here demonstrate increased expression of α-2,3 sialic acids compared to α-2,6 sialic acids in BeEFs, similar to sialylation profiles previously reported in bovine mammary tissue. These data also display increased viral fitness of the bovine origin HPAI H5N1 strains across bovine and avian cell lines, consistent with previous characterization in bovine mammary tissue. Furthermore, BeEFs were fully susceptible to a 2022 H1N1pdm09-like IAV strain while maintaining resistance to the 2009 H1N1pdm09 IAV as previously characterized in mammary cells. This study highlights the ongoing zoonotic adaptation of HPAI H5N1 in mammals and the potential for coinfection with select human H1N1 2009 pandemic lineage strains, enabling the potential development of reassortant strains. These data support the ability of BeEFs to serve as a complementary in vitro system for studying IAV infections in bovine hosts. IMPORTANCEZoonotic spillover to humans with avian influenza A subtypes, such as H5N1, can have extraordinarily high mortality rates. Recently, highly pathogenic avian influenza (HPAI) H5N1 has spread to dairy cattle and caused widespread disease in over a thousand herds across the United States. This widespread infection not only poses notable economic challenges to agricultural industries but also represents a notable concern to human public health. While studies of infection dynamics of HPAI H5N1 in cattle remain crucial, animal handling restrictions and a lack of well-characterized cell culture models make this work challenging. The significance of our research lies in identifying an in vitro system—primary bovine embryonic fibroblasts (BeEFs)—as a physiologically relevant in vitro system for studying these infection dynamics, helping to mitigate the limitations imposed by stringent animal handling requirements.

Supplementary Fig. S3 shows analysis of lung tumor burden and tumor cell proliferation/death in the Kras; p53 model with Keap1/Nrf2 mutation.

Supplementary Fig. S2 shows lung tumor analysis data for Keap1/Nrf2 mutant mice in the KrasG12D/+ model.

Supplementary Fig. S5 shows lung tumor analysis data in the KrasG12D/+; p53fl/fl model with single copy Nrf2 deletion.

Carbohydrate sulfation plays a pivotal role in modulating the strength of Siglec-glycan interactions. Recently, new aspects of Siglec binding to sulfated cell surface carbohydrates have been discovered, but the class of glycan presenting these sulfated Siglec ligands has not been fully elucidated. In this study, the contribution of different classes of glycans to cis and trans Siglec ligands was investigated within cells expressing the carbohydrate sulfotransferase 1 (CHST1) or CHST2. For some Siglecs, the glycan class mediating binding was clear, such as O-glycans for Siglec-7 and N-glycans for Siglec-2 and Siglec-9. Both N-glycans and mucin-type O-glycans contributed to ligands for Siglec-3, -5, -8, and -15. However, significant levels of Siglec-3 and -8 ligands remained in CHST1-expressing cells lacking complex N-glycans and mucin-type O-glycans. A combination of genetic, pharmacological, and enzymatic treatment strategies ruled out heparan sulfates and glycoRNA as contributors, although Siglec-8 did exhibit some binding to glycolipids. Genetic disruption of O-mannose glycans within CHST1-expressing cells had a small but significant impact on Siglec-3 and -8 binding, demonstrating that this class of glycans can present sulfated Siglec ligands. We also investigated the ability of sulfated cis ligands to mask Siglec-3 and Siglec-7. For Siglec-7, cis ligands were again found to be mucin-type O-glycans. While N-glycans were the major sulfated trans ligands for Siglec-3, disruption of complex mucin-type O-glycans had the largest impact on Siglec-3 masking. Overall, this study enhances our knowledge of the types of sulfated glycans that can serve as Siglec ligands.

Supplementary Fig. S4 shows the H-scores by tumor grade of Nrf2 and Nqo1 immunohistochemical staining in KrasG12D/+; p53fl/fl and KrasG12D/+; p53fl/fl; Keap1R554Q/R554Q mouse models with single copy Nrf2 deletion.

BRAF V600E-inhibition effectively treats melanoma, but acquired resistance rapidly develops. Protein expression profiles, mitochondrial energetics, metabolomics and fluxomics data in cell line, xenograft, and patient-derived xenograft systems revealed that concerted reprogramming of metabolic pathways (including glutaminolysis, glycolysis, TCA cycle, electron transport chain [ETC], and transsulfuration), along with an immediate cytoprotective response to drug-induced oxidative stress, underpins drug-tolerant persister cancer cell survival. Realignment of cysteine (Cys) metabolism, in particular an immediate upregulation of cystathionine-γ-lyase (CSE), was vital in persister cells. The oxidative cellular environment, drug-induced elevated cystine uptake and oxidative Cys catabolism, increased intracellular cystine/Cys ratios, thereby favoring cystine as a CSE substrate. This produces persulfides and hydrogen sulfide to protect protein thiols and support elevated energy demand in persister cells. Combining BRAF V600E inhibitors with CSE inhibitors effectively diminished proliferative relapse in culture models and increased progression-free survival of xenografted mice. This, together with induced CSE expression in patient samples under BRAF-V600E-inhibition, reveals an approach to increase BRAF-V600E-targeted therapeutic efficacy.

Supplementary Fig. S1 shows that the CA-Keap1R554Q allele does not exhibit hypomorphism in mouse embryonic fibroblasts.

The emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions as well as limited availability of established cell culture models. Primary Bovine Embryonic Fibroblasts (BeEFs) were collected from a Montana cow and are investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles, infectious virus production, viral replication, and plaque morphology in both BeEFs and chicken DF-1 cells following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here show increased viral fitness of the bovine origin HPAI H5N1 strains across multiple species and bovine susceptibility to human seasonal IAV. This study highlights the ability of BeEFs to serve as a model for studying IAV infections in bovine hosts.

AbstractIt has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

Glycans are emerging as important regulators of T cell function but remain poorly characterized across the functionally distinct populations that exist in vivo . Here, we couple single-cell analysis technologies with soluble lectins and chemical probes to interrogate glycosylation patterns on major T cell populations across multiple mouse and human tissues. Our analysis focused on terminal glycan epitopes with immunomodulatory functions, including sialoglycan ligands for Siglecs. We demonstrate that glycosylation patterns are diverse across the resting murine T cell repertoire and dynamically remodelled in response to antigen-specific stimulation. Surprisingly, we find that human T cell populations do not share the same glycoprofiles or glycan remodelling dynamics as their murine counterparts. We show that these differences can be explained by divergent regulation of glycan biosynthesis pathways between the species. These results highlight fundamental glycophysiological differences between mouse and human T cells and reveal features that are critical to consider for glycan-targeted therapies.

Cell surface complex carbohydrates, known as glycans, are positioned to be the first point of contact between two cells. Indeed, interactions between glycans with glycan-binding can modulate cell-cell interactions. This concept is particularly relevant for immune cells, which use an array of glycan-binding proteins to help in the process of differentiating ‘self’ from ‘non-self’. This is exemplified by the sialic acid-binding immunoglobulin-type lectins (Siglecs), which recognize sialic acid. Given that sialic acid is relatively unique to vertebrates, immune cells leverage Siglecs to recognize sialic acid as a marker of ‘self’. Siglecs serve many biological roles, with most of these functions regulated through interactions with their sialoglycan ligands. In this review, we provide a comprehensive update on the ligands of Siglecs and how Siglec-sialoglycan interactions help regulate immune cells in the adaptive and innate immune system.

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using alpha-2,3- and alpha-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.

Glycans constitute a significant fraction of biomolecular diversity on the surface of cells across all the species in all kingdoms of life. As the structure of glycans is not encoded by the DNA of the host organisms, it is impossible to use cutting-edge DNA technology to study the role of cellular glycosylation or to understand how cell-surface glycome is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a genetically-encoded liquid glycan array (LiGA) platform that allows profiling of glycan:GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing (NGS). LiGA is a library of DNA-barcoded bacteriophages coated with 5-1500 copies of a glycan; the DNA barcode inside each bacteriophage encodes the structure and density of the displayed glycans. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs displayed on the surface of such cells. This protocol provides detailed instructions of using LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI-TOF MS, the assembly of a LiGA library, and its deep-sequencing. Using the protocol detailed in this report, we measure a glycan-binding profile of the immunomodulatory SiglecLJ1, -2, -6, -7, and -9 expressed on the surface of different cell types and uncover previously unknown environment-dependent recognition of glycans by Siglec-receptors on the surface of live cells. Protocols similar to the one described in this report will make it possible to measure the precise glycan-binding profile of any GPBs displayed on the surface of any cell types.

Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages—liquid lectin array (LiLA)—detect subtle differences in density of glycans on cells. LiLA constructs displaying 73 copies of diCBM40 (CBM) lectin per virion (φ-CBM73) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. A high-valency φ-CBM290 display, or soluble CBM protein, cannot amplify the subtle differences detected by φ-CBM73. Similarly, multivalent displays of CBM and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer. Multivalent display of lectins offer in situ detection of minor differences in glycocalyx in cells both in vitro and in vivo not feasible to currently available technologies.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

Cysteine is required for synthesis of glutathione (GSH), coenzyme A, other sulfur-containing metabolites, and most proteins. In most cells, cysteine comes from extracellular disulfide sources including cystine, glutathione-disulfide, and peptides. The thioredoxin reductase-1 (TrxR1)- or glutathione-disulfide reductase (GSR)-driven enzymatic systems can fuel cystine reduction via thioredoxins, glutaredoxins, or other thioredoxin-fold proteins. Free cystine enters cells thorough the cystine-glutamate antiporter, xCT, but systemically, plasma glutathione-disulfide might predominate as a cystine source. Erastin, inhibiting both xCT and voltage-dependent anion channels, induces ferroptotic cell death, so named because this type of cell death is antagonized by iron-chelators. Many cancer cells seem to be predisposed to ferroptosis, which has been proposed as a targetable cancer liability. Ferroptosis is associated with lipid peroxidation and loss of either glutathione peroxidase-4 (GPX4) or ferroptosis suppressor protein-1 (FSP1), which each prevent accumulation of lipid peroxides. It has been suggested that an xCT inhibition-induced cellular cysteine-deficiency lowers GSH levels, starving GPX4 for reducing power and allowing membrane lipid peroxides to accumulate, thereby causing ferroptosis. Aspects of ferroptosis are however not fully understood and need to be further scrutinized, for example that neither disruption of GSH synthesis, loss of GSH, nor disruption of glutathione disulfide reductase (GSR), triggers ferroptosis in animal models. Here we reevaluate the relationships between Erastin, xCT, GPX4, cellular cysteine and GSH, RSL3 or ML162, and ferroptosis. We conclude that, whereas both Cys and ferroptosis are potential liabilities in cancer, their relationship to each other remains insufficiently understood.

Cancer cells adjust the composition of their glycocalyx to increase its thickness and create a physical barrier that shields them from immune recognition and engagement.