Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle.

Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.

Comparison of intestinal absorption of soybean protein isolate-, glutenin- and peanut protein isolate-bound Nε-(carboxymethyl) lysine after in vitro gastrointestinal digestion.

Advanced glycation end products (AGEs), a heterogeneous compound existed in processed foods, are related to chronic diseases when they are accumulated excessively in human organs. Protein-bound Nε-(carboxymethyl) lysine (CML) as a typical AGE, is widely determined to evaluate AGEs level in foods and in vivo. This study investigated the intestinal absorption of three protein-bound CML originated from main food raw materials (soybean, wheat and peanut). After in vitro gastrointestinal digestion, the three protein-bound CML digests were ultrafiltered and divided into four fractions: less than 1 kDa, between 1 and 3 kDa, between 3 and 5 kDa, greater than 5 kDa. Caco-2 cell monolayer model was further used to evaluate the intestinal absorption of these components. Results showed that the absorption rates of soybean protein isolate (SPI)-, glutenin (Glu)-, peanut protein isolate (PPI)-bound CML were 30.18%, 31.57% and 29.5%, respectively. The absorption rates of components with MW less than 5 kDa accounted for 19.91% (SPI-bound CML), 22.59% (Glu-bound CML), 23.64% (PPI-bound CML), respectively, and these samples were absorbed by paracellular route, transcytosis route and active route via PepT-1. Taken together, these findings demonstrated that all three protein-bound CML digests with different MW can be absorbed in diverse absorption pathways by Caco-2 cell monolayer model. This research provided a theoretical basis for scientific evaluation of digestion and absorption of AGEs in food.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane.

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Influence of chirality and sequence in lysine-rich lipopeptide biosurfactants and micellar model colloid systems.

Lipopeptides can self-assemble into diverse nanostructures which can be programmed to incorporate peptide sequences to achieve a remarkable range of bioactivities. Here, the influence of peptide sequence and chirality on micelle structure and interactions is investigated in a series of lipopeptides bearing two lysine or D-lysine residues and tyrosine or tryptophan residues, attached to a hexadecyl lipid chain. All molecules self-assemble into micelles above a critical micelle concentration (CMC). Small-angle x-ray scattering (SAXS) is used to probe micelle shape and structure from the form factor and to probe inter-micellar interactions via analysis of structure factor. The CMC is obtained consistently from surface tension and electrical conductivity measurements. We introduce a method to obtain the zeta potential from the SAXS structure factor which is in good agreement with directly measured values. Atomistic molecular dynamics simulations provide insights into molecular packing and conformation within the lipopeptide micelles which constitute model self-assembling colloidal systems and biomaterials.

Rapid Revealing of Quorum Sensing (QS)-Regulated PLA, Biofilm and Lysine Targets of Lactiplantibacillus plantarum L3.

Quorum sensing (QS) can regulate the production of multiple functional factors in bacteria, but the process of identifying its regulatory targets is very complex and labor-intensive. In this study, an efficient and rapid method to find QS targets through prediction was used. The genome of Lactiplantibacillus plantarum (L. plantarum) L3 was sequenced and characterized, and then linked the L. plantarum L3 genome to the STRING database for QS system regulatory target prediction. A total of 3,167,484 base pairs (bps) were examined from the genome of L. plantarum L3, and 30 QS-related genes were discovered (including luxS). The STRING database prediction indicated that the 30 QS-related genes are mainly involved in the regulation of nine metabolic pathways. Furthermore, metE, metK, aroB, cysE, and birA1 were predicted to be regulatory targets of the LuxS/AI-2 QS system, and these five targets were validated based on quantitative real-time PCR and content determination. Successful elucidation of the LuxS/AI-2 QS system's key targets and regulation mechanism in L. plantarum L3 demonstrated the effectiveness of the new approach for predicting QS targets and provides a scientific basis for future work on improving regulation of functional factor production.

Lactate promotes microglial scar formation and facilitates locomotor function recovery by enhancing histone H4 lysine 12 lactylation after spinal cord injury.

Lactate-derived histone lactylation is involved in multiple pathological processes through transcriptional regulation. The role of lactate-derived histone lactylation in the repair of spinal cord injury (SCI) remains unclear. Here we report that overall lactate levels and lactylation are upregulated in the spinal cord after SCI. Notably, H4K12la was significantly elevated in the microglia of the injured spinal cord, whereas exogenous lactate treatment further elevated H4K12la in microglia after SCI. Functionally, lactate treatment promoted microglial proliferation, scar formation, axon regeneration, and locomotor function recovery after SCI. Mechanically, lactate-mediated H4K12la elevation promoted PD-1 transcription in microglia, thereby facilitating SCI repair. Furthermore, a series of rescue experiments confirmed that a PD-1 inhibitor or microglia-specific AAV-sh-PD-1 significantly reversed the therapeutic effects of lactate following SCI. This study illustrates the function and mechanism of lactate/H4K12la/PD-1 signaling in microglia-mediated tissue repair and provides a novel target for SCI therapy.

The Many Faces of Hypusinated eIF5A: Cell Context-Specific Effects of the Hypusine Circuit and Implications for Human Health.

The unique amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is exclusively formed on the translational regulator eukaryotic initiation factor 5A (eIF5A) via a process coined hypusination. Hypusination is mediated by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH), and hypusinated eIF5A (eIF5AHyp) promotes translation elongation by alleviating ribosome pauses at amino acid motifs that cause structural constraints, and it also facilitates translation initiation and termination. Accordingly, eIF5AHyp has diverse biological functions that rely on translational control of its targets. Homozygous deletion of Eif5a, Dhps, or Dohh in mice leads to embryonic lethality, and heterozygous germline variants in EIF5A and biallelic variants in DHPS and DOHH are associated with rare inherited neurodevelopmental disorders, underscoring the importance of the hypusine circuit for embryonic and neuronal development. Given the pleiotropic effects of eIF5AHyp, a detailed understanding of the cell context-specific intrinsic roles of eIF5AHyp and of the chronic versus acute effects of eIF5AHyp inhibition is necessary to develop future strategies for eIF5AHyp-targeted therapy to treat various human health problems. Here, we review the most recent studies documenting the intrinsic roles of eIF5AHyp in different tissues/cell types under normal or pathophysiological conditions and discuss these unique aspects of eIF5AHyp-dependent translational control.

Alcohol- and Low-Iron Induced Changes in Antioxidant and Energy Metabolism Associated with Protein Lys Acetylation.

Understanding the role of iron in ethanol-derived hepatic stress could help elucidate the efficacy of dietary or clinical interventions designed to minimize liver damage from chronic alcohol consumption. We hypothesized that normal levels of iron are involved in ethanol-derived liver damage and reduced dietary iron intake would lower the damage caused by ethanol. We used a pair-fed mouse model utilizing basal Lieber-DeCarli liquid diets for 22 weeks to test this hypothesis. In our mouse model, chronic ethanol exposure led to mild hepatic stress possibly characteristic of early-stage alcoholic liver disease, seen as increases in liver-to-body weight ratios. Dietary iron restriction caused a slight decrease in non-heme iron and ferritin (FeRL) expression while it increased transferrin receptor 1 (TfR1) expression without changing ferroportin 1 (FPN1) expression. It also elevated protein lysine acetylation to a more significant level than in ethanol-fed mice under normal dietary iron conditions. Interestingly, iron restriction led to an additional reduction in nicotinamide adenine dinucleotide (NAD+) and NADH levels. Consistent with this observation, the major mitochondrial NAD+-dependent deacetylase, NAD-dependent deacetylase sirtuin-3 (SIRT3), expression was significantly reduced causing increased protein lysine acetylation in ethanol-fed mice at normal and low-iron conditions. In addition, the detection of superoxide dismutase 1 and 2 levels (SOD1 and SOD2) and oxidative phosphorylation (OXPHOS) complex activities allowed us to evaluate the changes in antioxidant and energy metabolism regulated by ethanol consumption at normal and low-iron conditions. We observed that the ethanol-fed mice had mild liver damage associated with reduced energy and antioxidant metabolism. On the other hand, iron restriction may exacerbate certain activities of ethanol further, such as increased protein lysine acetylation and reduced antioxidant metabolism. This metabolic change may prove a barrier to the effectiveness of dietary reduction of iron intake as a preventative measure in chronic alcohol consumption.

Computational study on the Maillard reactions of glucose and galactose with lysine.

CONTEXT: Milk has nutrient-rich but thermal sensitive matrix that undergoes varying degrees of Maillard reaction (MR) at heating conditions. The MR mainly occurs between lysine residues (Lys) and lactose composed of glucose (Glc) and galactose (Gal), which are abundantly sourced from dairy products. In the present study, the MRs of Glc and Gal with Lys at the initial and intermediate stages have been investigated theoretically using density functional theory (DFT) to simulate the gaseous and aqueous phases. Reaction mechanisms have been proposed, and relative energy changes of different steps were calculated according to the total mass balance. The calculations reveal that both Nα- and Nε-amine groups of Lys can react with the carbonyl functional group of Glc and Gal with the similar potential energy profiles, and Gal is more reactive than Glc. However, the barrier in Nε-channel is lower than in Nα-channel, indicating a faster reaction rate through the former channel compared with the latter. The 5-hydroxymethyl-2-furfural (HMF) and derivative are formed under 3-deoxysone route in the intermediate stage. The calculation results are helpful for proposing a reasonable MR mechanism and suggesting possible control methods of the MRs. METHODS: In this study, different levels of DFT calculations have been conducted to investigate the mechanisms and favorability of generating MR products in Glc-Lys and Gal-Lys models at initial and intermediate stages in the gaseous and aqueous conditions. In order to elucidate the molecular models from the perspectives of chemistry and geometry, DFT calculations were performed by the mean of B3LYP functional at basis sets of 6-311 + + G (d, p) and 6-311 + + G (2df, 2p) with optional solvation settings. To examine the solvation effect, the study further constructed models with solvent H2O and calculated in wB97XD functional with 6-31 + G (d) basis set. All computations were carried out Gaussian 09 suite of quantum chemistry software.

Triptolide induced spermatogenesis dysfunction via ferroptosis activation by promoting K63-linked GPX4 polyubiquitination in spermatocytes.

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.

Single-electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins.

The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover the biological roles of post-translational modifications. Photoreactive crosslinkers are powerful tools for investigating readers. However, existing methods usually employ synthetically challenging photoreactive warheads, and their high-energy intermediates generated upon irradiation, such as nitrene and carbene, may cause substantial non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and subsequently crosslinks to a conserved tryptophan inside the binding pocket through single-electron transfer under ultraviolet irradiation. The crosslinking relies on a protein-templated σ-π electron donor-acceptor interaction between sulfonium and indole, ensuring excellent site selectivity for tryptophan in the active site and orthogonality to other methyllysine readers. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking strategy could be extended to develop other types of microenvironment-dependent conjugations to site-specific tryptophan.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Transglutaminase-mediated proximity labeling of a specific Lys residue in a native IgG antibody.

The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule.

Bacterial protein acetylation: mechanisms, functions, and methods for study.

Lysine acetylation is an evolutionarily conserved protein modification that changes protein functions and plays an essential role in many cellular processes, such as central metabolism, transcriptional regulation, chemotaxis, and pathogen virulence. It can alter DNA binding, enzymatic activity, protein-protein interactions, protein stability, or protein localization. In prokaryotes, lysine acetylation occurs non-enzymatically and by the action of lysine acetyltransferases (KAT). In enzymatic acetylation, KAT transfers the acetyl group from acetyl-CoA (AcCoA) to the lysine side chain. In contrast, acetyl phosphate (AcP) is the acetyl donor of chemical acetylation. Regardless of the acetylation type, the removal of acetyl groups from acetyl lysines occurs only enzymatically by lysine deacetylases (KDAC). KATs are grouped into three main superfamilies based on their catalytic domain sequences and biochemical characteristics of catalysis. Specifically, members of the GNAT are found in eukaryotes and prokaryotes and have a core structural domain architecture. These enzymes can acetylate small molecules, metabolites, peptides, and proteins. This review presents current knowledge of acetylation mechanisms and functional implications in bacterial metabolism, pathogenicity, stress response, translation, and the emerging topic of protein acetylation in the gut microbiome. Additionally, the methods used to elucidate the biological significance of acetylation in bacteria, such as relative quantification and stoichiometry quantification, and the genetic code expansion tool (CGE), are reviewed.

Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule.

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5'-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

METTL14-mediated upregulation of lncRNA HOTAIR represses PP1α expression by promoting H3K4me1 demethylation in oxycodone-treated mice.

N6-methyladenosine (m6A) methylation is a vital epigenetic mechanism associated with drug addiction. However, the relationship between m6A modification and oxycodone rewarding is less well explored. Based on an open field test, the present study evaluated oxycodone rewarding using chromatin immunoprecipitation PCR, immunofluorescence, and RNA sequencing. A marked increase in METTL14 protein and a decrease in PP1α protein due to oxycodone abundance in the striatal neurons were observed in a dose- and time-dependent manner. Oxycodone markedly increased LSD1 expression, and decreased H3K4me1 expression in the striatum. In the open field test, intra-striatal injection of METTL14 siRNA, HOTAIR siRNA, or LSD1 shRNA blocked oxycodone-induced increase in locomotor activity. The downregulation of PP1α was also inhibited after treatment with METTL14/HOTAIR siRNA and LSD1 shRNA. Enhanced binding of LSD1 with CoRest and of CoRest with the PP1α gene induced by oxycodone was also reversed by LSD1 shRNA. In addition, H3K4me1 demethylation was also blocked by the treatment. In summary, the investigation confirmed that METTL14-mediated upregulation of HOTAIR resulted in the repression of PP1α, which in turn facilitated the recruitment of LSD1, thus catalyzing H3K4me1 demethylation and promoting oxycodone addiction.

[An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides].

Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In "shotgun" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.

Nanoparticles that Distinguish Chemical and Supramolecular Contexts of Lysine for Single-Site Functionalization of Protein.

Lysine is one of the most abundant residues on the surface of proteins and its site-selective functionalization is extremely challenging. The existing methods of functionalization rely on differential reactivities of lysine on a protein, making it impossible to label less reactive lysines selectively. We here report polymeric nanoparticles that mimic enzymes involved in the posttranslational modifications of proteins that distinguish the chemical and supramolecular contexts of a lysine and deliver the labeling reagent precisely to its ε amino group. The nanoparticles are prepared through molecular imprinting of cross-linkable surfactant micelles, plus an in situ, on-micelle derivatization of the peptide template prior to the imprinting. The procedures encode the polymeric nanoparticles with all the supramolecular information needed for sequence identification and precise labeling, allowing single-site functionalization of a predetermined lysine on the target protein in a mixture.

Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3.

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection.

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma.

BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.