Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged in Wuhan City, Hubei Province, China in December 2019. By Feb. 11, 2020, the World Health Organization (WHO) officially named the disease resulting from infection with SARS-CoV-2 as coronavirus disease 2019 (COVID-19). COVID-19 represents a spectrum of clinical manifestations that typically include fever, dry cough, and fatigue, often with pulmonary involvement. SARS-CoV-2 is highly contagious and most individuals within the population at large are susceptible to infection. Wild animal hosts and infected patients are currently the main sources of disease which is transmitted via respiratory droplets and direct contact. Since the outbreak, the Chinese government and scientific community have acted rapidly to identify the causative agent and promptly shared the viral gene sequence, and have carried out measures to contain the epidemic. Meanwhile, recent research has revealed critical aspects of SARS-CoV-2 biology and disease pathogenesis; other studies have focused on epidemiology, clinical features, diagnosis, management, as well as drug and vaccine development. This review aims to summarize the latest research findings and to provide expert consensus. We will also share ongoing efforts and experience in China, which may provide insight on how to contain the epidemic and improve our understanding of this emerging infectious disease, together with updated guidance for prevention, control, and critical management of this pandemic.
Amino Acid Motifs ; Animals ; Antiviral Agents ; Betacoronavirus ; genetics ; China ; epidemiology ; Communicable Disease Control ; methods ; Coronavirus Infections ; diagnosis ; epidemiology ; physiopathology ; prevention & control ; therapy ; Humans ; Immunization, Passive ; Medicine, Chinese Traditional ; Pandemics ; Pneumonia, Viral ; diagnosis ; epidemiology ; physiopathology ; therapy ; Protein Domains ; Spike Glycoprotein, Coronavirus ; chemistry ; Viral Vaccines

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Amino Acid Motifs ; Animals ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; HEK293 Cells ; Humans ; Integrin beta3 ; genetics ; metabolism ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Signal Transduction

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

Amino Acid Motifs ; DEAD-box RNA Helicases ; chemistry ; Humans ; Protein Domains ; Structure-Activity Relationship

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.
Amino Acid Sequence ; Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Chromosomal Proteins, Non-Histone ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Crystallography, X-Ray ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Histones ; chemistry ; genetics ; metabolism ; Models, Molecular ; Oryza ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Isoforms ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Viridiplantae ; genetics ; metabolism

Amino Acid Motifs ; Bacterial Proteins ; chemistry ; Crystallography, X-Ray ; Klebsiella pneumoniae ; chemistry ; Protein Domains ; Transcription Factors ; chemistry

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.
Amino Acid Motifs ; Binding Sites ; Cell Cycle Proteins ; chemistry ; Crystallography, X-Ray ; Humans ; Multienzyme Complexes ; chemistry ; Protein Phosphatase 2 ; chemistry ; Protein Structure, Quaternary ; Protein-Serine-Threonine Kinases ; chemistry

Viral infection induces numerous tripartite motif (TRIM) proteins to control antiviral immune signaling and viral replication. Particularly, SPRY-containing TRIM proteins are found only in vertebrates and they control target protein degradation by their RING-finger and SPRY domains, and proper cytoplasmic localization. To understand TRIM30 function, we analyzed its localization pattern and putative roles of its RING-finger and SPRY domains. We found that TRIM30 is located in actin-mediated cytoplasmic bodies and produces colocalized ubiquitin chains in SPRY domain- and RING-finger domain-dependent ways that are degraded by autophagy and the proteasome. These results suggest a TRIM protein-dependent degradation mechanism by cytoplasmic body formation with actin networks.
Amino Acid Sequence ; Animals ; Autophagy ; Cell Line ; Inclusion Bodies/*metabolism ; Intracellular Signaling Peptides and Proteins/chemistry/genetics/*metabolism ; Mice ; Molecular Sequence Data ; Polyubiquitin/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Proteolysis ; RING Finger Domains

As a protein originally found in plant pathogenic bacteria, transcription activator-like effectors (TALEs) can be fused with the cleaving domain of restriction endonuclease (For example Fok I) to form artificial nucleases named TALENs. These proteins are dependent on variable numbers of tandem Repeats of TALEs to recognize and bind DNA sequences. Each of these repeats consists of a set of approximately 34 amino acids, composed of about 32 conserved amino acids and 2 highly variable amino acids called repeat variant di-residues (RVDs). RVDs distinguish one TALE from another and can make TALEs have a simple cipher for the one-to-one recognition for proteins and DNA bases. Based on this, in theory, artificially constructed TALENs could recognize and break DNA sites specifically and arbitrarily to perform gene knockout, insertion or modification. We reviewed the development of this technology in multi-level and multi species, and its advantages and disadvantages compared with ZFNs and CRISPR/Cas technology. We also address its special advantages in industrial microbe breeding, vector construction, targeting precision, high efficiency of editing and biological safety.
Amino Acid Motifs ; Biotechnology ; DNA ; chemistry ; Endonucleases ; chemistry ; Tandem Repeat Sequences ; Trans-Activators ; chemistry