Lysine Residues Research Articles

Using in vivo intact structure for system-wide quantitative analysis of changes in proteins

Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimer’s disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions.

Histone Deacetylases in Metabolism: The Known and the Unexplored.

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from key lysine residues on histone and non-histone proteins and thereby regulate gene transcription. They have been implicated in several biological processes in both healthy and pathologic settings. This review discusses the role of HDACs in multiple metabolically active tissues and highlights their contribution to the pathogenesis of tissue-specific maladaptation and diseases. We also summarize the current knowledge gaps and potential ways to address them in future studies.

Discovery and Characterization of BAY-184: A New Potent and Selective Acylsulfonamide-Benzofuran In Vivo-Active KAT6AB Inhibitor.

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

The key to 2,6-dichloro-1,4-benzoquinone reproductive toxicity and green tea detoxification: Covalent binding and competitive binding

Halobenzoquinones (HBQs) are ubiquitous disinfection by-products (DBPs) in chlorinated drinking water with various health risks including reproductive toxicity, while the potential mechanisms are still unclear. Although green tea exhibits common detoxifying properties, its ability to mitigate the toxicity of HBQs still needs to be further deepened and explored. This study attempted to investigate the possible mechanism of the most common HBQ, 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) induced reproductive toxicity and elucidate the protective effect of green tea using a series of liquid chromatography-tandem mass spectrometry (LC-MS) approaches. Firstly, in vivo experiments showed that 2,6-DCBQ could induce testicular damage in male rats via significantly decreasing sperm-associated Leydig cells and seminiferous tubules. Then, in vitro incubation of 2,6-DCBQ with amino acids suggested that 2,6-DCBQ could bind to proteins via residues of cysteine or lysine and provided five additional modification patterns. Following, proteomics analysis revealed that at least 42 proteins were modified by 2,6-DCBQ, which were mainly enriched in the reproductive system. These results highlighted the significance of covalent protein modification in 2,6-DCBQ reproductive toxicity. Fortunately, we found that catechins (a class of major components of green tea) could competitively bind to 2,6-DCBQ in vivo and in vitro, reducing the amount and type of 2,6-DCBQ-protein adducts, thereby attenuating the reproductive system damage caused by 2,6-DCBQ. This study provides new insights into 2,6-DCBQ-induced reproductive system damage and reveals a new mechanism of green tea detoxification. Moreover, these findings offer potential strategies for alleviating the harmful impacts of environmental toxicants on human health.

Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of N-Propargylglycine.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ1-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

PreMLS: The undersampling technique based on ClusterCentroids to predict multiple lysine sites.

The translated protein undergoes a specific modification process, which involves the formation of covalent bonds on lysine residues and the attachment of small chemical moieties. The protein's fundamental physicochemical properties undergo a significant alteration. The change significantly alters the proteins' 3D structure and activity, enabling them to modulate key physiological processes. The modulation encompasses inhibiting cancer cell growth, delaying ovarian aging, regulating metabolic diseases, and ameliorating depression. Consequently, the identification and comprehension of post-translational lysine modifications hold substantial value in the realms of biological research and drug development. Post-translational modifications (PTMs) at lysine (K) sites are among the most common protein modifications. However, research on K-PTMs has been largely centered on identifying individual modification types, with a relative scarcity of balanced data analysis techniques. In this study, a classification system is developed for the prediction of concurrent multiple modifications at a single lysine residue. Initially, a well-established multi-label position-specific triad amino acid propensity algorithm is utilized for feature encoding. Subsequently, PreMLS: a novel ClusterCentroids undersampling algorithm based on MiniBatchKmeans was introduced to eliminate redundant or similar major class samples, thereby mitigating the issue of class imbalance. A convolutional neural network architecture was specifically constructed for the analysis of biological sequences to predict multiple lysine modification sites. The model, evaluated through five-fold cross-validation and independent testing, was found to significantly outperform existing models such as iMul-kSite and predML-Site. The results presented here aid in prioritizing potential lysine modification sites, facilitating subsequent biological assays and advancing pharmaceutical research. To enhance accessibility, an open-access predictive script has been crafted for the multi-label predictive model developed in this study.

Acetylation-Dependent Compaction of the Histone H4 Tail Ensemble.

Acetylation of the histone H4 tail (H4Kac) has been established as a significant regulator of chromatin architecture and accessibility; however, the molecular mechanisms that underlie these observations remain elusive. Here, we characterize the ensemble features of the histone H4 tail and determine how they change following acetylation on specific sets of lysine residues. Our comprehensive account is enabled by a robust combination of experimental and computational biophysical methods that converge on molecular details including conformer size, intramolecular contacts, and secondary structure propensity. We find that acetylation significantly alters the chemical environment of basic patch residues (16-20) and leads to tail compaction that is partially mediated by transient intramolecular contacts established between the basic patch and N-terminal amino acids. Beyond acetylation, we identify that the protonation state of H18, which is affected by the acetylation state, is a critical regulator of ensemble characteristics, highlighting the potential for interplay between the sequence context and post-translational modifications to define the ensemble features of intrinsically disordered regions. This study elucidates molecular details that could link H4Kac with the regulation of chromatin architecture, illuminating a small piece of the complex network of molecular mechanisms underlying the histone code hypothesis.

Stoichiometry of ligand binding and role of C-terminal lysines in Mycobacterium tuberculosis and human GAPDH multifunctionality.

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; EC1.2.1.12) has several functions in Mycobacterium tuberculosis (Mtb) and the human host. Apart from its role in glycolysis, it serves both as a cell surface and a secreted receptor for plasmin(ogen) (Plg/Plm), transferrin (Tf), and lactoferrin (Lf). Plg sequestration by Mtb GAPDH facilitates bacterial adhesion and tissue invasion, while an equivalent interaction with host GAPDH regulates immune cell migration. In both, host and microbe, internalization of Tf/Lf-GAPDH complexes serves as a route for iron acquisition. To date, the structure of Mtb GAPDH or the residues involved in these moonlighting interactions have not been identified. This study provides the first known X-ray crystal structure of Mtb GAPDH. Through further mutagenesis and functional assays, we found that the C-terminal lysines of Mtb and human GAPDH affect enzyme activity and ligand binding. We also establish the stoichiometry of Plg, Tf and Lf interactions with the GAPDH tetramer. Lastly, molecular simulation studies reveal the interactions of the C-terminal lysine residues.

Acetylation of DnaJ facilitates the proliferation of BmNPV by affecting the transport of nucleocapsids

DnaJ (Orf40), a late-expressed factor of Bombyx mori nucleopolyhedrovirus (BmNPV), is essential for the budding of virions and influences the transfer of the nucleocapsid from the nucleus to the cytoplasm. Previous studies showed that the knockdown of dnaj could prevent the nucleocapsid from exiting the nucleus, but the underlying regulatory mechanism remains unknown. In our previous acetylomic analysis of BmN cells infected with wild-type BmNPV, we found that a lysine residue (K17) was acetylated 36h post-infection, and the acetylation level of this site was upregulated about 3.5-fold. Here, we found that deacetylation of DnaJ K17 significantly inhibited viral proliferation without affecting viral DNA replication. Furthermore, deacetylation of DnaJ K17 affected the interaction with two nucleocapsid-associated proteins, Ac66 and VP80, which in turn affected the production of nucleocapsids, as well as their transport within the nucleus along F-actin fibers, leading to a decrease in the export of nucleocapsids from the nucleus. The reduced amount of nucleocapsids in the cytoplasm ultimately led to a decrease in the production of budded virions and consequently inhibited viral proliferation. In conclusion, acetylation of DnaJ affects nucleocapsid production and transport, thereby influencing viral proliferation.

Disrupting CENP-N mediated SEPT9 methylation as a strategy to inhibit aerobic glycolysis and liver metastasis in colorectal cancer.

Colorectal cancer (CRC) is a prevalent malignancy with a high mortality rate, primarily due to liver metastasis. This study explores the role of centromere protein N (CENP-N) in mediating the methylation of septin 9 (SEPT9) and its subsequent effects on aerobic glycolysis and liver metastasis in CRC. We employed in vitro and in vivo experiments, including single-cell RNA sequencing, methylation-specific PCR (MSP), ChIP assays, and various functional assays to assess the impact of CENP-N and SEPT9 on CRC cell proliferation, migration, invasion, and metabolic reprogramming. Our data reveal that CENP-N directly interacts with SEPT9, enhancing its methylation at specific lysine residues. This modification significantly upregulates key glycolytic enzymes, thereby promoting aerobic glycolysis, CRC cell proliferation, and migration. In vivo studies further demonstrate that the CENP-N/SEPT9 axis facilitates liver metastasis of CRC, as confirmed by fluorescence imaging and histological analysis. This study identifies a novel pathway where CENP-N-mediated methylation of SEPT9 drives metabolic reprogramming and metastasis in CRC. These findings suggest potential therapeutic targets for inhibiting CRC progression and liver metastasis, offering new insights into CRC pathogenesis.

A vector system encoding histone H3 mutants facilitates manipulations of the neuronal epigenome

The differentiation of developmental cell lineages is associated with genome-wide modifications in histone H3 methylation. However, the causal role of histone H3 methylation in transcriptional regulation and cell differentiation has been difficult to test in mammals. The experimental overexpression of histone H3 mutants carrying lysine-to-methionine (K-to-M) substitutions has emerged as an alternative tool for inhibiting the endogenous levels of histone H3 methylation at specific lysine residues. Here, we leverage the use of histone K-to-M mutants by creating Enhanced Episomal Vectors that enable the simultaneous depletion of multiple levels of histone H3 lysine 4 (H3K4) or lysine 9 (H3K9) methylation in projection neurons of the mouse cerebral cortex. Our approach also facilitates the simultaneous depletion of H3K9 and H3K27 trimethylation (H3K9me3 and H3K27me3, respectively) in cortical neurons. In addition, we report a tamoxifen-inducible Cre-FLEX system that allows the activation of mutant histones at specific developmental time points or in the adult cortex, leading to the depletion of specific histone marks. The tools presented here can be implemented in other experimental systems, such as human in vitro models, to test the combinatorial role of histone methylations in developmental fate decisions and the maintenance of cell identity.

Oxygen Glucose Deprivation-Induced Lactylation of H3K9 Contributes to M1 Polarization and Inflammation of Microglia Through TNF Pathway.

Hypoxia-induced M1 polarization of microglia and resultant inflammation take part in the damage caused by hypoxic-ischemic encephalopathy (HIE). Histone lactylation, a novel epigenetic modification where lactate is added to lysine residues, may play a role in HIE pathogenesis. This study investigates the role of histone lactylation in hypoxia-induced M1 microglial polarization and inflammation, aiming to provide insights for HIE treatment. In this study, we assessed the effects of hypoxia on microglial polarization using both an HIE animal model and an oxygen-glucose deprivation cell model. Histone lactylation at various lysine residues was detected by Western blotting. Microglial polarization and inflammatory cytokines were analyzed by immunofluorescence, qPCR, and Western blotting. RNA sequencing, ChIP-qPCR, and siRNA were used to elucidate mechanisms of H3K9 lactylation. H3K9 lactylation increased due to cytoplasmic lactate during M1 polarization. Inhibiting P300 or reducing lactate dehydrogenase A expression decreased H3K9 lactylation, suppressing M1 polarization. Transcriptomic analysis indicated that H3K9 lactylation regulated M1 polarization via the TNF signaling pathway. ChIP-qPCR confirmed H3K9 lactylation enrichment at the TNFα locus, promoting OGD-induced M1 polarization and inflammation. H3K9 lactylation promotes M1 polarization and inflammation via the TNF pathway, identifying it as a potential therapeutic target for neonatal HIE.

UBC18 E2 conjugating enzyme depends on SINAT1 E3 ligase to destabilize the ESCRT component FREE1 in plant iron deficiency responses.

E2 ubiquitin-conjugating enzymes play a crucial role in the ubiquitination process by catalyzing ubiquitin transfer. Although the function of ubiquitin-protein ligases (E3s) in plants response to diverse abiotic stress by targeting specific substrates has been well studied, the involvement of E2s in environmental responses and their downstream targets are not well understood. In this study, we demonstrated that the E2 ubiquitin-conjugating enzyme 18 (UBC18) influences the stability of FREE1 to modulate iron deficiency stress. UBC18 affects the ubiquitination of FREE1 and promotes its degradation, and overexpression of UBC18 decreases plants' sensitivity to iron deficiency by reducing FREE1 level, whereas the ubc18 mutant exhibits sensitivity due to elevated FREE1 accumulation. This study also identified that lysine residues K227, K295, K315, and K540 are required for FREE1 ubiquitination and stability regulation. Mutating these lysine residues in FREE1 resulted in plants' sensitivity to iron starvation. Taken together, our findings shed light on the mechanism of UBC18 in responding to iron deficiency stress by modulating the abundance of FREE1, and further elucidate the role of ubiquitination sites in FREE1 stability regulation and the plant iron deficiency response.

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

DNA polymerase η is regulated by mutually exclusive mono-ubiquitination and mono-NEDDylation.

DNA polymerase eta (Pol η) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein (PIP) motifs within Pol η, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol η itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol η can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol η is mono-NEDDylated under normal conditions, these levels rapidly increase by inhibiting the COP9 signalosome, suggesting that mono-NEDDylation is maintained under strong negative regulation. Finally, we provide data to support that mono-ubiquitination is important for Pol η foci formation and suggest that NEDDylation disrupts this process. These results reveal a new mechanism of Pol η regulation by ubiquitin-like proteins.

Incorporation of pyridoxal-5′-phosphate into the apoenzyme: A structural study of D-amino acid transaminase from Haliscomenobacter hydrossis

Pyridoxal-5′-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from Haliscomenobacter hydrossis. PLP binding to the apoenzyme was slow in buffer, but was accelerated in the presence of substrates. Two crystal structures of the apoenzyme were obtained: the directly obtained apoenzyme (PDB ID: 7P8O) and the one obtained by soaking crystals of the holoenzyme in a phenylhydrazine solution (PDB ID: 8YRU). The mechanism of PLP association with the apoenzyme was proposed on the basis of structural analysis of these apo forms. Three rearrangement steps, including (I) anchoring of the PLP via the phosphate group, (II) displacement of two loops, and (III) Schiff-bonding between the PLP and the ε-amino group of the catalytic lysine residue, reconstituted the active holo form of the transaminase from H. hydrossis. The results obtained allowed us to determine in the active site a permanent part and elements that are assembled by PLP, these findings may be useful for transaminase engineering for biocatalysis.

Acetylation of Histone H3 in Cancer Progression and Prognosis

Cancer is a multifactorial disease resulting from both genetic factors and epigenetic changes. Histone acetylation, a post-translational modification, which alters chromatin architecture and regulates gene expression is associated with cancer initiation, development and progression. Aberrations in global histone acetylation levels are observed in various cancer cells and are also associated with patients’ tumor aggressiveness. Therefore, histone acetylation may have prognostic utility and serve as a potential biomarker of cancer progression and patients’ prognosis. The reversible modification of histones by an acetyl group is versatile. One particular histone can be acetylated on different lysine residues, subsequently resulting in different biological outcomes. Here, we discuss recent findings on the acetylation of the highly conserved histone protein H3 in the context of cancer biology. Specifically, we review the acetylation of particular H3 residues in various cancer types. We further highlight the significance of H3 acetylation levels as a potential cancer biomarker with prognostic implications.

Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping.

Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.

Lysine residues are not required for proteasome-mediated proteolysis of cellular prion protein

Cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrPC is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrPC degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrPC are involved in the ubiquitination and subsequent degradation of PrPC. We generated a plasmid vector expressing a mutant PrPC (called lysine-null PrPC) in which all lysine residues were replaced with arginine residues. Subsequently, we established stably transformed cell lines (designated HpL2-1 PrP-WT and HpL2-1 PrP-K/R, respectively) using the mouse PrPC-deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrPC (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrPC which translocated efficiently to the plasma membrane. Subsequently, using immunoblotting and confocal microscopy, we found that treatment with cycloheximide (CHX; a protein synthesis inhibitor) significantly reduced PrPC expression in both these transformed cell lines, indicating that WT and lysine-null PrPC are degraded similarly. Taken together, these results indicate that the lysine residues of PrPC do not regulate its degradation.

Structural analysis of the CJ0600 protein from Campylobacter jejuni

The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5′-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported.