MetaDegron: multimodal feature-integrated protein language model for predicting E3 ligase targeted degrons.

Protein degradation through the ubiquitin proteasome system at the spatial and temporal regulation is essential for many cellular processes. E3 ligases and degradation signals (degrons), the sequences they recognize in the target proteins, are key parts of the ubiquitin-mediated proteolysis, and their interactions determine the degradation specificity and maintain cellular homeostasis. To date, only a limited number of targeted degron instances have been identified, and their properties are not yet fully characterized. To tackle on this challenge, here we develop a novel deep-learning framework, namely MetaDegron, for predicting E3 ligase targeted degron by integrating the protein language model and comprehensive featurization strategies. Through extensive evaluations using benchmark datasets and comparison with existing method, such as Degpred, we demonstrate the superior performance of MetaDegron. Among functional features, MetaDegron allows batch prediction of targeted degrons of 21 E3 ligases, and provides functional annotations and visualization of multiple degron-related structural and physicochemical features. MetaDegron is freely available at http://modinfor.com/MetaDegron/. We anticipate that MetaDegron will serve as a useful tool for the clinical and translational community to elucidate the mechanisms of regulation of protein homeostasis, cancer research, and drug development.

DegronMD: Leveraging Evolutionary and Structural Features for Deciphering Protein-Targeted Degradation, Mutations, and Drug Response to Degrons.

Protein-targeted degradation is an emerging and promising therapeutic approach. The specificity of degradation and the maintenance of cellular homeostasis are determined by the interactions between E3 ubiquitin ligase and degradation signals, known as degrons. The human genome encodes over 600 E3 ligases; however, only a small number of targeted degron instances have been identified so far. In this study, we introduced DegronMD, an open knowledgebase designed for the investigation of degrons, their associated dysfunctional events, and drug responses. We revealed that degrons are evolutionarily conserved and tend to occur near the sites of protein translational modifications, particularly in the regions of disordered structure and higher solvent accessibility. Through pattern recognition and machine learning techniques, we constructed the degrome landscape across the human proteome, yielding over 18,000 new degrons for targeted protein degradation. Furthermore, dysfunction of degrons disrupts the degradation process and leads to the abnormal accumulation of proteins; this process is associated with various types of human cancers. Based on the estimated phenotypic changes induced by somatic mutations, we systematically quantified and assessed the impact of mutations on degron function in pan-cancers; these results helped to build a global mutational map on human degrome, including 89,318 actionable mutations that may induce the dysfunction of degrons and disrupt protein degradation pathways. Multiomics integrative analysis unveiled over 400 drug resistance events associated with the mutations in functional degrons. DegronMD, accessible at https://bioinfo.uth.edu/degronmd, is a useful resource to explore the biological mechanisms, infer protein degradation, and assist with drug discovery and design on degrons.

Effect of acupuncture on regulating IL-17, TNF-ɑ and AQPs in Sjögren's syndrome.

AIM: The aim of the study was to observe the effect of acupuncture on regulating interleukin (IL)-17, tumor necrosis factor (TNF)-ɑ, and aquaporins (AQPs) in Sjögren's syndrome (SS) on patients and on non-obese diabetic (NOD) models. METHODS: Levels of anti-AQP 1, 5, 8, and 9 antibodies, IL-17, and TNF-ɑ in the serum of SS patients were compared prior and following 20 acupuncture treatment visits during 8 weeks. While in murine model, five groups were divided to receive interventions for 4 weeks, including control, model, acupuncture, isoflurane, and hydroxychloroquine. The submaxillofacial gland index, histology, immunohistochemistry of AQP1, 5, salivary flow, together with IL-17, and TNF-ɑ expression in peripheral blood were compared among the groups. RESULTS: Acupuncture reduced IL-17, TNF-ɑ, and immunoglobin A levels, and numeric analog scale of dryness in 14 patients with SS (p < 0.05). The salivary flow was increased, and the water intake decreased in NOD mice receiving acupuncture treatments. IL-17 and TNF-ɑ levels in peripheral serum were down-regulated (p < 0.05) and AQP1, 5 expression in the submandibular glands up-regulated in mice. CONCLUSION: The effect on relieving xerostomia with acupuncture may be achieved by up-regulating the expression of AQP1. AQP5, down-regulating levels of IL-17 and TNF-ɑ, and a decrease in inflammation of glands.

Elevated MCU Expression by CaMKIIδB Limits Pathological Cardiac Remodeling.

BACKGROUND: Calcium (Ca2+) is a key regulator of energy metabolism. Impaired Ca2+ homeostasis damages mitochondria, causing cardiomyocyte death, pathological hypertrophy, and heart failure. This study investigates the regulation and the role of the mitochondrial Ca2+ uniporter (MCU) in chronic stress-induced pathological cardiac remodeling. METHODS: MCU knockout or transgenic mice were infused with isoproterenol (ISO; 10 mg/kg per day, 4 weeks). Cardiac hypertrophy and remodeling were evaluated by echocardiography and histology. Primary cultured rodent adult cardiomyocytes were treated with ISO (1 nmol/L, 48 hours). Intracellular Ca2+ handling and cell death pathways were monitored. Adenovirus-mediated gene manipulations were used in vitro. RESULTS: Chronic administration of the ß-adrenergic receptor agonist ISO increased the levels of the MCU and the MCU complex in cardiac mitochondria, raising mitochondrial Ca2+ concentrations, in vivo and in vitro. ISO also upregulated MCU without affecting its regulatory proteins in adult cardiomyocytes. It is interesting that ISO-induced cardiac hypertrophy, fibrosis, contractile dysfunction, and cardiomyocyte death were exacerbated in global MCU knockout mice. Cardiomyocytes from knockout mice or overexpressing a dominant negative MCU exhibited defective intracellular Ca2+ handling and activation of multiple cell death pathways. Conversely, cardiac-specific overexpression of MCU maintained intracellular Ca2+ homeostasis and contractility, suppressed cell death, and prevented ISO-induced heart hypertrophy. ISO upregulated MCU expression through activation of Ca2+/calmodulin kinase II δB (CaMKIIδB) and promotion of its nuclear translocation via calcineurin-mediated dephosphorylation at serine 332. Nuclear CaMKIIδB phosphorylated CREB (cAMP-response element binding protein), which bound the Mcu promoter to enhance Mcu gene transcription. CONCLUSIONS: The ß-adrenergic receptor/CaMKIIδB/CREB pathway upregulates Mcu gene expression in the heart. MCU upregulation is a compensatory mechanism that counteracts stress-induced pathological cardiac remodeling by preserving Ca2+ homeostasis and cardiomyocyte viability.

Deep4mC: systematic assessment and computational prediction for DNA N4-methylcytosine sites by deep learning.

DNA N4-methylcytosine (4mC) modification represents a novel epigenetic regulation. It involves in various cellular processes, including DNA replication, cell cycle and gene expression, among others. In addition to experimental identification of 4mC sites, in silico prediction of 4mC sites in the genome has emerged as an alternative and promising approach. In this study, we first reviewed the current progress in the computational prediction of 4mC sites and systematically evaluated the predictive capacity of eight conventional machine learning algorithms as well as 12 feature types commonly used in previous studies in six species. Using a representative benchmark dataset, we investigated the contribution of feature selection and stacking approach to the model construction, and found that feature optimization and proper reinforcement learning could improve the performance. We next recollected newly added 4mC sites in the six species' genomes and developed a novel deep learning-based 4mC site predictor, namely Deep4mC. Deep4mC applies convolutional neural networks with four representative features. For species with small numbers of samples, we extended our deep learning framework with a bootstrapping method. Our evaluation indicated that Deep4mC could obtain high accuracy and robust performance with the average area under curve (AUC) values greater than 0.9 in all species (range: 0.9005-0.9722). In comparison, Deep4mC achieved an AUC value improvement from 10.14 to 46.21% when compared to previous tools in these six species. A user-friendly web server (https://bioinfo.uth.edu/Deep4mC) was built for predicting putative 4mC sites in a genome.

mUSP: a high-accuracy map of the in situ crosstalk of ubiquitylation and SUMOylation proteome predicted via the feature enhancement approach.

Reversible post-translational modification (PTM) orchestrates various biological processes by changing the properties of proteins. Since many proteins are multiply modified by PTMs, identification of PTM crosstalk site has emerged to be an intriguing topic and attracted much attention. In this study, we systematically deciphered the in situ crosstalk of ubiquitylation and SUMOylation that co-occurs on the same lysine residue. We first collected 3363 ubiquitylation-SUMOylation (UBS) crosstalk site on 1302 proteins and then investigated the prime sequence motifs, the local evolutionary degree and the distribution of structural annotations at the residue and sequence levels between the UBS crosstalk and the single modification sites. Given the properties of UBS crosstalk sites, we thus developed the mUSP classifier to predict UBS crosstalk site by integrating different types of features with two-step feature optimization by recursive feature elimination approach. By using various cross-validations, the mUSP model achieved an average area under the curve (AUC) value of 0.8416, indicating its promising accuracy and robustness. By comparison, the mUSP has significantly better performance with the improvement of 38.41 and 51.48% AUC values compared to the cross-results by the previous single predictor. The mUSP was implemented as a web server available at http://bioinfo.ncu.edu.cn/mUSP/index.html to facilitate the query of our high-accuracy UBS crosstalk results for experimental design and validation.

Alkali-Amide-Catalyzed One-Pot Aminoallylation of Aldehydes with Allylbenzenes.

The deprotonation of allylbenzene was successfully demonstrated with a catalytic alkali amide base (NaN(SiMe3)2). The deprotonated allyl anion could be trapped by in situ generated N-(trimethylsilyl) aldimines to provide value-added homoallylic amines (39 examples, 68-98% yields) in a one-pot manner with excellent liner selectivity. Compared with the previously reported method for the synthesis of homoallylic amines, this method does not need to use the preinstalled protection groups on the imines, which need to be removed after the reaction to obtain the N-H free homoallylic amine derivatives.

KinaseMD: kinase mutations and drug response database.

Mutations in kinases are abundant and critical to study signaling pathways and regulatory roles in human disease, especially in cancer. Somatic mutations in kinase genes can affect drug treatment, both sensitivity and resistance, to clinically used kinase inhibitors. Here, we present a newly constructed database, KinaseMD (kinase mutations and drug response), to structurally and functionally annotate kinase mutations. KinaseMD integrates 679 374 somatic mutations, 251 522 network-rewiring events, and 390 460 drug response records curated from various sources for 547 kinases. We uniquely annotate the mutations and kinase inhibitor response in four types of protein substructures (gatekeeper, A-loop, G-loop and αC-helix) that are linked to kinase inhibitor resistance in literature. In addition, we annotate functional mutations that may rewire kinase regulatory network and report four phosphorylation signals (gain, loss, up-regulation and down-regulation). Overall, KinaseMD provides the most updated information on mutations, unique annotations of drug response especially drug resistance and functional sites of kinases. KinaseMD is accessible at https://bioinfo.uth.edu/kmd/, having functions for searching, browsing and downloading data. To our knowledge, there has been no systematic annotation of these structural mutations linking to kinase inhibitor response. In summary, KinaseMD is a centralized database for kinase mutations and drug response.

Miniature Fourier transform spectrometer based on a fiber-tip interferometer.

The miniaturization of spectrometers have attracted much attention owning to the demand for portable or in situ spectral analysis in a wide variety of fields, but it is a great challenge to push them into practical applications due to high cost, complicated configuration, and sensitivity to external disturbance. We report on a miniature Fourier transform (FT) spectrometer based on fiber-tip Fizeau interferometer. Hand pulling or any other types of force can be used to drive optical path difference (OPD) scan. Interferences are monitored as a function of time by two photodetectors, one is used to detect the whole interferogram while the other to measure single-wavelength interferogram. In this design, the instantaneous interference intensity as well as OPD can be obtained in an accurate way so that the exact spatial interferogram of the incident spectrum can be worked out. Consequently, the incident spectrum can be retrieved by FT method. A resolution of 7.69 cm-1 in the wavelength range of 1400 nm ∼ 1700 nm is achieved. Experimental results show that the performance of our device is comparable to the commercial benchtop spectrometer. Our device is independent of the complicated fabrication procedures, easy of usage, and cost effective. We envision that the proposed design will inspire a new concept for constructing simple and cheap spectrometers that is well suited for practical applications.

Micro-opto-electro-mechanical systems accelerometer based on the Talbot effect of double-layer diffraction gratings.

In this paper, we present the design, fabrication, and test of a micro-opto-electro-mechanical systems (MOEMS) accelerometer based on the Talbot effect of double-layer diffraction gratings. The detection of acceleration is realized by using the highly sensitive displacement characteristic of Talbot imaging of near-field diffraction with double-layer gratings. For the purpose of obtaining optimal contrast of the optical interferometric detection, the parameters of the gratings are optimized by the finite-difference time-domain (FDTD) simulation. The experimental results indicate that this MOEMS accelerometer with the proposed design can achieve a resolution of 246 µg, sensitivity of 6.1 V/g, and bias stability of 0.02 mg. The proposed accelerometer can be operated at higher accelerations (∼80g), which shows significant potential for being used in applications that require detection of strong and fast vibrations, especially in vibration sensing of vehicles and geophysical seismic sensing in real time.

Elevated EZH2 in ischemic heart disease epigenetically mediates suppression of NaV1.5 expression.

Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism. METHODS: In this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR. FINDINGS: The median age of our cohort was 73·5 years (range 42-84; IQR 67·5-77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue. INTERPRETATION: The primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination. FUNDING: None.

6mA-Finder: a novel online tool for predicting DNA N6-methyladenine sites in genomes.

MOTIVATION: DNA N6-methyladenine (6 mA) has recently been found as an essential epigenetic modification, playing its roles in a variety of cellular processes. The abnormal status of DNA 6 mA modification has been reported in cancer and other disease. The annotation of 6 mA marks in genome is the first crucial step to explore the underlying molecular mechanisms including its regulatory roles. RESULTS: We present a novel online DNA 6 mA site tool, 6 mA-Finder, by incorporating seven sequence-derived information and three physicochemical-based features through recursive feature elimination strategy. Our multiple cross-validations indicate the promising accuracy and robustness of our model. 6 mA-Finder outperforms its peer tools in general and species-specific 6 mA site prediction, suggesting it can provide a useful resource for further experimental investigation of DNA 6 mA modification. AVAILABILITY AND IMPLEMENTATION: https://bioinfo.uth.edu/6mA_Finder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A case of endocrine mucin-producing sweat gland carcinoma with distant metastasis.

Endocrine mucin-producing sweat gland carcinoma (EMPSGC) is a rare cutaneous adnexal neoplasm typically arising on the face of older individuals, most commonly around the eyelids. Histopathologic features include a circumscribed proliferation of low-grade epithelioid cells with areas of cystic and cribriform growth, foci of intracytoplasmic and extracellular mucin, and coexpression of endocrine, neuroendocrine, and cytokeratin markers by immunohistochemistry. Given histopathologic and immunohistochemical similarities, EMPSGC is often likened to solid papillary carcinoma of the breast and endocrine ductal carcinoma in situ, and is thought by many to represent a forme fruste of mucinous carcinoma of the skin. To date, the vast majority of reported cases of EMPSGC have been described as having indolent behavior, with no cases of distant metastasis yet reported. Here we report a unique case of EMPSGC that recurred over several years following standard surgical excision and Mohs micrographic surgery, with subsequent metastasis to the parotid gland and axial skeleton.

iEKPD 2.0: an update with rich annotations for eukaryotic protein kinases, protein phosphatases and proteins containing phosphoprotein-binding domains.

Here, we described the updated database iEKPD 2.0 (http://iekpd.biocuckoo.org) for eukaryotic protein kinases (PKs), protein phosphatases (PPs) and proteins containing phosphoprotein-binding domains (PPBDs), which are key molecules responsible for phosphorylation-dependent signalling networks and participate in the regulation of almost all biological processes and pathways. In total, iEKPD 2.0 contained 197 348 phosphorylation regulators, including 109 912 PKs, 23 294 PPs and 68 748 PPBD-containing proteins in 164 eukaryotic species. In particular, we provided rich annotations for the regulators of eight model organisms, especially humans, by compiling and integrating the knowledge from 100 widely used public databases that cover 13 aspects, including cancer mutations, genetic variations, disease-associated information, mRNA expression, DNA & RNA elements, DNA methylation, molecular interactions, drug-target relations, protein 3D structures, post-translational modifications, protein expressions/proteomics, subcellular localizations and protein functional annotations. Compared with our previously developed EKPD 1.0 (∼0.5 GB), iEKPD 2.0 contains ∼99.8 GB of data with an ∼200-fold increase in data volume. We anticipate that iEKPD 2.0 represents a more useful resource for further study of phosphorylation regulators.

Imaging features of fungal pneumonia in haematopoietic stem cell transplant patients.

Patients who have received haematopoietic stem cell transplantation (HSCT) have a high rate of pulmonary complications, and in this immunosuppressed population, fungal pneumonia is of great concern. Fungal pneumonia can have a similar appearance to non-infectious pulmonary processes in HSCT patients, and radiologists should be familiar with the subtle features that may help to differentiate these disease entities. The focus of this article is on the diagnosis of fungal pneumonia in HSCT patients with an emphasis on radiologists' roles in establishing the diagnosis of fungal pneumonia and the guidance of clinical management.

A small-molecule LF3 abrogates ß-catenin/TCF4-mediated suppression of NaV1.5 expression in HL-1 cardiomyocytes.

Increased nuclear ß-catenin interacting with T-cell factor 4 (TCF4) affects the expression of target genes including SCN5A in ischemic heart disease, which is characterized by frequent ventricular tachycardia/fibrillation. A complex of ß-catenin and TCF4 inhibits cardiac Na+ channel activity by reducing NaV1.5 expression through suppressing SCN5A promoter activity in HL-1 cardiomyocytes. LF3, a 4-thioureido-benzenesulfonamide derivative and an inhibitor of ß-catenin/TCF4 interaction, has been shown to block the self-renewal capacity of cancer stem cells. We performed studies to determine if LF3 can reverse suppressive effects of ß-catenin/TCF4 signaling on the expression of NaV1.5 in HL-1 cardiomyocytes. Western blotting and real-time qRT-PCR analyses showed that 10 µM LF3 significantly increased the expression of NaV1.5 but it did not alter ß-catenin and TCF4 expression. Subcellular fractionation analysis demonstrated that LF3 significantly increased the levels of NaV1.5 in both membrane and cytoplasm. Whole-cell patch-clamp recordings revealed that Na+ currents were significantly increased with no changes in the steady-state parameters, activation and inactivation time constants and recovery from inactivation of Na+ channel in HL-1 cells treated with LF3. Immunoprecipitation exhibited that LF3 blocked the interaction of ß-catenin and TCF4. Luciferase reporter assays performed in HEK 293 cells and HL-1 revealed that LF3 increased the SCN5A promoter activity in HL-1 cells and prevented ß-catenin suppressive effect on SCN5A promoter activity in HEK 293 cells. Taken together, we conclude that LF3, an inhibitor of ß-catenin/TCF4 interaction, elevates NaV1.5 expression, leading to increase Na+ channel activity in HL-1 cardiomyocytes.

Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development.

Bone morphogenetic protein (BMP) and Wnt pathways regulate cell proliferation and differentiation, but how these two pathways interact and mediate their nuclear actions in the heart, especially during late cardiac development, remains poorly defined. T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors, including Lef1, Tcf7, Tcf7l1, and Tcf7l2, are important nuclear mediators of canonical Wnt/ß-catenin signaling throughout cardiac development. We reveal that these TCF/LEF family members direct heart maturation through distinct temporal and spatial control. TCF7 and LEF1 decrease while TCF7L1 and TCF7L2 remain relatively stable during heart development. LEF1 is mainly expressed in mesenchymal cells in valvular regions. TCF7 and TCF7L1 are detected in the nucleus of mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. Tcf7l2 is the primary TCF/LEF family member in cardiomyocytes and undergoes alternative splicing during heart development. A TCF7L2 intensity gradient opposite to that of ß-catenin and cardiomyocyte proliferative activity is present in fetal hearts. Wnt activation by cardiac deletion of APC, a negative Wnt regulator, dramatically increases Cyclin D2 and Bmp4 expression. BMP signal transducing transcription factors, the mothers against decapentaplegic homologs (SMADs) are increasingly phosphorylated upon Wnt activation. LEF1/TCF7 displaces TCF7L2 and cooperates with pSMAD 1/5/8 in the regulatory elements of Cyclin D2 and Bmp4 promoters to promote ß-catenin recruitment and transcriptional activation. Finally, we demonstrate that TCF7L2 is a transcriptional suppressor of Cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments.

Propylselen inhibits cancer cell growth by targeting glutamate dehydrogenase at the NADP+ binding site.

High levels of glutamate dehydrogenase (GDH) activity are associated with hypoglycemia, cancer, and Parkinson's disease. Propylselen was synthesized to investigate its mechanism of GDH inhibition in comparison with Ebselen and Epigallocatechin gallate (EGCG). Because Ebselen was found to crosslink with the peptide (AA299-341) at the active site of E.coli GDH, the Cys, Pro, and Lys residues of the corresponding peptide were mutagenized to Ala residues. Using enzyme kinetics and biomolecular interaction assays, we found that the conserved GDH P320 residue is important for propylselen binding, C321 for Ebselen binding, and K341 for EGCG binding. In addition, these 3 mutations abolished NADP+ binding to E. coli GDH in the absence of glutamate substrate, but in the presence of glutamate, the catalytic activity of the mutants was reduced only by 2-4 fold, indicating that a substrate-induced fit mechanism exists in E. coli GDH. Furthermore, biochemical analysis showed that NADP+ had high affinity (Kd of 77 nM) for GDH; by targeting the NADP binding site, propylselen effectively inhibited both E. coli and human GDH activity and improved anticancer activity.

Site-Specific Systematic Analysis of Lysine Modification Crosstalk.

Research has revealed that post-translational modifications (PTMs) that occur at lysine (PLMs) can cooperatively regulate various biological processes by crosstalk. However, the trend of the crosstalk between multiple PLMs and the properties of PLM crosstalk require additional investigation. Here, the crosstalk among acetylation, succinylation, and SUMOylation is systematically studied in a site-specific waz. First, crosstalk between SUMOylation is detected and succinylation is found to be underexpressed, whereas succinylation tends to crosstalk with acetylation and SUMOylation on the same lysine residue while PLM crosstalk is tissue-specific across different species. Further analysis reveals that different PLMs tend to occur crosstalk at diverse subcellular compartments and structural regions, and they participate in distinct biological processes and functions. Additionally, short-term evolutionary analysis shows that there is no additional evolutionary pressure on PLMs crosstalk sites, as found by comparison with singly modified sites. Finally, phylogenetic classification reveals that genes with co-occupied lysine crosstalk are more likely to have higher evolutionary similarity and possess a tendency to cluster in the specific branch. The integrated approach reported here has the potential for large-scale prioritization of in situ crosstalk of PLM candidates and provides a profound understanding of the underlying relationship between different lysine modifications.