ABSTRACT
SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Repair , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Models, Molecular , Molecular Structure , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Synthetic Lethal MutationsABSTRACT
Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.
Subject(s)
Binding Sites , Molecular Docking Simulation , Molecular Dynamics Simulation , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acids , Antibody Affinity , Epitopes/chemistry , Epitopes/metabolism , Humans , Multiprotein Complexes/chemistry , Mutagenesis , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.
Subject(s)
COVID-19/physiopathology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/immunology , COVID-19 Vaccines/immunology , Cathepsins/metabolism , ErbB Receptors/antagonists & inhibitors , Humans , Immunization Schedule , Immunization, Secondary , Phytotherapy/methods , Plants, Medicinal , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , Protein Structural Elements/physiology , Spike Glycoprotein, Coronavirus/metabolism , Viral Protease Inhibitors/pharmacology , Viral Protease Inhibitors/therapeutic useABSTRACT
Voltage-gated calcium channels (VGCCs) are widely expressed in the brain, heart and vessels, smooth and skeletal muscle, as well as in endocrine cells. VGCCs mediate gene transcription, synaptic and neuronal structural plasticity, muscle contraction, the release of hormones and neurotransmitters, and membrane excitability. Therefore, it is not surprising that VGCC dysfunction results in severe pathologies, such as cardiovascular conditions, neurological and psychiatric disorders, altered glycemic levels, and abnormal smooth muscle tone. The latest research findings and clinical evidence increasingly show the critical role played by VGCCs in autism spectrum disorders, Parkinson's disease, drug addiction, pain, and epilepsy. These findings outline the importance of developing selective calcium channel inhibitors and modulators to treat such prevailing conditions of the central nervous system. Several small molecules inhibiting calcium channels are currently used in clinical practice to successfully treat pain and cardiovascular conditions. However, the limited palette of molecules available and the emerging extent of VGCC pathophysiology require the development of additional drugs targeting these channels. Here, we provide an overview of the role of calcium channels in neurological disorders and discuss possible strategies to generate novel therapeutics.
Subject(s)
Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Animals , Calcium Channel Agonists/therapeutic use , Calcium Channel Blockers/therapeutic use , Calcium Channels/chemistry , Calcium Channels/classification , Calcium Channels/genetics , Clinical Studies as Topic , Disease Management , Disease Susceptibility , Drug Discovery , Drug Evaluation, Preclinical , Humans , Ligands , Nervous System Diseases/diagnosis , Nervous System Diseases/drug therapy , Nervous System Diseases/etiology , Nervous System Diseases/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Treatment OutcomeABSTRACT
Before entering the cell, the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. Hence, this RBD is a critical target for the development of antiviral agents. Recent studies have discovered that SARS-CoV-2 variants with mutations in the RBD have spread globally. The purpose of this in silico study was to determine the potential of a fruit bromelain-derived peptide. DYGAVNEVK. to inhibit the entry of various SARS-CoV-2 variants into human cells by targeting the hACE binding site within the RBD. Molecular docking analysis revealed that DYGAVNEVK interacts with several critical RBD binding residues responsible for the adhesion of the RBD to hACE2. Moreover, 100 ns MD simulations revealed stable interactions between DYGAVNEVK and RBD variants derived from the trajectory of root-mean-square deviation (RMSD), radius of gyration (Rg), and root-mean-square fluctuation (RMSF) analysis, as well as free binding energy calculations. Overall, our computational results indicate that DYGAVNEVK warrants further investigation as a candidate for preventing SARS-CoV-2 due to its interaction with the RBD of SARS-CoV-2 variants.
Subject(s)
Angiotensin-Converting Enzyme 2 , Bromelains , Computer Simulation , Protein Interaction Domains and Motifs , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Bromelains/chemistry , Bromelains/pharmacology , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/pharmacology , Protein Binding , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , COVID-19 Drug TreatmentABSTRACT
Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, ß, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small ß-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.
Subject(s)
Black Widow Spider/chemistry , Calcium/chemistry , Neurotoxins/chemistry , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Spider Venoms/chemistry , Animals , Binding Sites , Black Widow Spider/pathogenicity , Calcium/metabolism , Cloning, Molecular , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Ion Transport , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Potentials/physiology , Models, Molecular , Neurotoxins/genetics , Neurotoxins/metabolism , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spider Venoms/genetics , Spider Venoms/metabolismABSTRACT
Erianin (Eri) is the extract of Dendrobium chrysotoxum Lindl. The NLRP3 inflammasome is a multiprotein complex that plays key roles in a wide variety of chronic inflammation-driven human diseases. Nevertheless, little is known about the protection of Eri against NLRP3 inflammasome-related diseases. In this study, we demonstrated that Eri inhibited NLRP3 inflammasome activation in vitro and in vivo. Mechanistically, Eri directly interacted with NLRP3, leading to inhibition of NLRP3 inflammasome assembly. Eri associated with the Walker A motif in the NACHT domain and suppressed NLRP3 ATPase activity. In mouse models, Eri had therapeutic effects on peritonitis, gouty arthritis and type 2 diabetes, via NLRP3. More importantly, Eri was active ex vivo for synovial fluid cells and monocytes from patients with IAV infection and gout. Eri may serve as a potential novel therapeutic compound against NLRP3-driven diseases.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Gouty/drug therapy , Bibenzyls/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Inflammasomes/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Peritonitis/drug therapy , Phenol/pharmacology , Animals , Arthritis, Gouty/genetics , Arthritis, Gouty/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Disease Models, Animal , Dogs , HEK293 Cells , Humans , Inflammasomes/genetics , Inflammasomes/metabolism , Madin Darby Canine Kidney Cells , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Peritonitis/genetics , Peritonitis/metabolism , Protein Interaction Domains and Motifs , THP-1 CellsABSTRACT
Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.
Subject(s)
Bacterial Proteins/chemistry , Coliphages/genetics , DNA Restriction-Modification Enzymes/chemistry , Escherichia coli/genetics , Escherichia/genetics , Plasmids/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Coliphages/metabolism , Crystallography, X-Ray , DNA Restriction-Modification Enzymes/genetics , DNA Restriction-Modification Enzymes/metabolism , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/metabolism , Escherichia/metabolism , Escherichia/virology , Escherichia coli/metabolism , Escherichia coli/virology , Gene Expression , Genomic Islands , Genomics/methods , Models, Molecular , Plasmids/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of â¼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.
Subject(s)
DNA-Binding Proteins/metabolism , Neurodegenerative Diseases/pathology , Peptides/pharmacology , Small Molecule Libraries/pharmacology , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/physiology , Binding Sites , Computational Biology/methods , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Drug Evaluation, Preclinical/methods , Humans , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Peptides/chemistry , Protein Interaction Domains and Motifs , Transcription Factors/chemistry , Tumor Suppressor Protein p53/chemistryABSTRACT
Since the start of the outbreak of coronavirus disease 2019 in Wuhan, China, there have been more than 150 million confirmed cases of the disease reported to the World Health Organization. The beta variant (B.1.351 lineage), the mutation lineages of SARS-CoV-2, had increase transmissibility and resistance to neutralizing antibodies due to multiple mutations in the spike protein. N501Y, K417N and E484K, in the receptor binding domain (RBD) region may induce a conformational change of the spike protein and subsequently increase the infectivity of the beta variant. The L452R mutation in the epsilon variant (the B.1.427/B.1.429 variants) also reduced neutralizing activity of monoclonal antibodies. In this study, we discovered that 300 µg/mL GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit the binding between ACE2 and wild-type (Wuhan type) RBD spike protein. GB-2 can inhibit the binding between ACE2 and RBD with K417N-E484K-N501Y mutation in a dose-dependent manner. GB-2 inhibited the binding between ACE2 and the RBD with a single mutation (K417N or N501Y or L452R) except the E484K mutation. In the compositions of GB-2, glycyrrhiza uralensis Fisch. ex DC., theaflavin and (+)-catechin cannot inhibit the binding between ACE2 and wild-type RBD spike protein. Theaflavin 3-gallate can inhibit the binding between ACE2 and wild-type RBD spike protein. Our results suggest that GB-2 could be a potential candidate for the prophylaxis of some SARS-CoV-2 variants infection in the further clinical study because of its inhibition of binding between ACE2 and RBD with K417N-E484K-N501Y mutations or L452R mutation.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Biflavonoids/pharmacology , COVID-19 , Catechin/pharmacology , Gallic Acid/analogs & derivatives , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing/immunology , Antioxidants/pharmacology , Antiviral Agents/pharmacology , COVID-19/immunology , COVID-19/virology , Drug Discovery , Gallic Acid/pharmacology , HEK293 Cells , Humans , Medicine, East Asian Traditional , Mutation , Protein Binding/physiology , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
As a key enzyme regulating postprandial blood glucose, α-Glucosidase is considered to be an effective target for the treatment of diabetes mellitus. In this study, a simple, rapid, and effective method for enzyme inhibitors screening assay was established based on α-glucosidase catalyzes reactions in a personal glucose meter (PGM). α-glucosidase catalyzes the hydrolysis of maltose to produce glucose, which triggers the reduction of ferricyanide (K3[Fe(CN)6]) to ferrocyanide (K4[Fe(CN)6]) and generates the PGM detectable signals. When the α-glucosidase inhibitor (such as acarbose) is added, the yield of glucose and the readout of PGM decreased accordingly. This method can achieve the direct determination of α-glucosidase activity by the PGM as simple as the blood glucose tests. Under the optimal experimental conditions, the developed method was applied to evaluate the inhibitory activity of thirty-four small-molecule compounds and eighteen medicinal plants extracts on α-glucosidase. The results exhibit that lithospermic acid (52.5 ± 3.0%) and protocatechualdehyde (36.8 ± 2.8%) have higher inhibitory activity than that of positive control acarbose (31.5 ± 2.5%) at the same final concentration of 5.0 mM. Besides, the lemon extract has a good inhibitory effect on α-glucosidase with a percentage of inhibition of 43.3 ± 3.5%. Finally, the binding sites and modes of four active small-molecule compounds to α-glucosidase were investigated by molecular docking analysis. These results indicate that the PGM method is feasible to screening inhibitors from natural products with simple and rapid operations.
Subject(s)
Benzaldehydes/pharmacology , Benzofurans/pharmacology , Blood Glucose/analysis , Catechols/pharmacology , Depsides/pharmacology , Diabetes Mellitus, Type 2/diagnosis , Glycoside Hydrolase Inhibitors/pharmacology , Monitoring, Ambulatory/methods , alpha-Glucosidases/blood , Acarbose/chemistry , Acarbose/pharmacology , Benzaldehydes/chemistry , Benzaldehydes/isolation & purification , Benzofurans/chemistry , Benzofurans/isolation & purification , Binding Sites , Biosensing Techniques/instrumentation , Catechols/chemistry , Catechols/isolation & purification , Depsides/chemistry , Depsides/isolation & purification , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Glycoside Hydrolase Inhibitors/chemistry , Humans , Hydrolysis , Kinetics , Maltose/metabolism , Molecular Docking Simulation , Monitoring, Ambulatory/instrumentation , Plant Extracts/chemistry , Plants, Medicinal , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thermodynamics , Wearable Electronic Devices , alpha-Glucosidases/chemistryABSTRACT
Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer's disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.
Subject(s)
Cell Membrane/metabolism , Disulfides/metabolism , Neurons/metabolism , tau Proteins/metabolism , Animals , Cell Line, Tumor , Cysteine , Disulfides/chemistry , Humans , Mice , Models, Molecular , Mutation , Protein Conformation, alpha-Helical , Protein Folding , Protein Interaction Domains and Motifs , Secretory Pathway , Structure-Activity Relationship , tau Proteins/chemistry , tau Proteins/geneticsABSTRACT
The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
Subject(s)
Kinetochores/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Protozoan Proteins/genetics , Structure-Activity Relationship , Trypanosoma brucei brucei/genetics , Zinc/metabolismABSTRACT
A newly-emergent beta-coronavirus, SARS-CoV-2, rapidly has become a pandemic since 2020. It is a serious respiratory disease and caused more than 100 million of deaths in the world. WHO named it COVIA-19 and there is no effective targeted drug for it. The main treatment strategies include chemical medicine, traditional Chinese medicine (TCM) and biologics. Due to SARS-CoV-2 uses the spike proteins (S proteins) on its envelope to infect human cells, monoclonal antibodies that neutralize the S protein have become one of the hot research areas in the current research and treatment of SARS-CoV-2. In this study, we reviewed the antibodies that have been reported to have neutralizing activity against the SARS-CoV-2 infection. According to their different binding epitope regions in RBD or NTD, they are classified, and the mechanism of the representative antibodies in each category is discussed in depth, which provides potential foundation for future antibody and vaccine therapy and the development of antibody cocktails against SARS-CoV-2 mutants.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , COVID-19/therapy , COVID-19 Vaccines/immunology , Epitopes/immunology , Humans , Models, Molecular , Neutralization Tests , Pandemics , Protein Interaction Domains and Motifs , Receptors, Virus/chemistry , SARS-CoV-2/genetics , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with ß subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.
Subject(s)
Potassium Channels/chemistry , Protein Interaction Domains and Motifs , Spectrum Analysis , Action Potentials , Animals , CHO Cells , Cricetulus , Humans , Ion Channel Gating , Models, Molecular , Molecular Conformation , Patch-Clamp Techniques , Potassium Channels/metabolism , Protein Binding , Spectrum Analysis/methods , Structure-Activity RelationshipABSTRACT
The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.
Subject(s)
2S Albumins, Plant/chemistry , Anti-Bacterial Agents/chemistry , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Moringa oleifera/chemistry , Mustard Plant/chemistry , 2S Albumins, Plant/isolation & purification , 2S Albumins, Plant/pharmacology , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/chemistry , Amidohydrolases/genetics , Amidohydrolases/metabolism , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Binding Sites , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/growth & development , Gram-Positive Bacteria/enzymology , Gram-Positive Bacteria/growth & development , Microbial Sensitivity Tests , Molecular Docking Simulation , Plant Extracts/chemistry , Plant Leaves/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and MotifsABSTRACT
Jellyfish venom is well known for its local skin toxicities and various lethal accidents. The main symptoms of local jellyfish envenomation include skin lesions, burning, prickling, stinging pain, red, brown, or purplish tracks on the skin, itching, and swelling, leading to dermonecrosis and scar formation. However, the molecular mechanism behind the action of jellyfish venom on human skin cells is rarely understood. In the present study, we have treated the human HaCaT keratinocyte with Nemopilema nomurai jellyfish venom (NnV) to study detailed mechanisms of actions behind the skin symptoms after jellyfish envenomation. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS), cellular changes at proteome level were examined. The treatment of NnV resulted in the decrease of HaCaT cell viability in a concentration-dependent manner. Using NnV (at IC50), the proteome level alterations were determined at 12 h and 24 h after the venom treatment. Briefly, 70 protein spots with significant quantitative changes were picked from the gels for MALDI-TOF/MS. In total, 44 differentially abundant proteins were successfully identified, among which 19 proteins were increased, whereas 25 proteins were decreased in the abundance levels comparing with their respective control spots. DAPs involved in cell survival and development (e.g., Plasminogen, Vinculin, EMILIN-1, Basonuclin2, Focal adhesion kinase 1, FAM83B, Peroxisome proliferator-activated receptor-gamma co-activator 1-alpha) decreased their expression, whereas stress or immune response-related proteins (e.g., Toll-like receptor 4, Aminopeptidase N, MKL/Myocardin-like protein 1, hypoxia up-regulated protein 1, Heat shock protein 105 kDa, Ephrin type-A receptor 1, with some protease (or peptidase) enzymes) were up-regulated. In conclusion, the present findings may exhibit some possible key players during skin damage and suggest therapeutic strategies for preventing jellyfish envenomation.
Subject(s)
Cnidarian Venoms/toxicity , Keratinocytes/drug effects , Proteins/metabolism , Scyphozoa , Animals , Bites and Stings/metabolism , Cell Line , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Humans , Keratinocytes/metabolism , Protein Interaction Domains and Motifs , Proteomics , Skin/drug effects , Skin/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The novel SARS-CoV-2 uses ACE2 (Angiotensin-Converting Enzyme 2) receptor as an entry point. Insights on S protein receptor-binding domain (RBD) interaction with ACE2 receptor and drug repurposing has accelerated drug discovery for the novel SARS-CoV-2 infection. Finding small molecule binding sites in S protein and ACE2 interface is crucial in search of effective drugs to prevent viral entry. In this study, we employed molecular dynamics simulations in mixed solvents together with virtual screening to identify small molecules that could be potential inhibitors of S protein -ACE2 interaction. Observation of organic probe molecule localization during the simulations revealed multiple sites at the S protein surface related to small molecule, antibody, and ACE2 binding. In addition, a novel conformation of the S protein was discovered that could be stabilized by small molecules to inhibit attachment to ACE2. The most promising binding site on RBD-ACE2 interface was targeted with virtual screening and top-ranked compounds (DB08248, DB02651, DB03714, and DB14826) are suggested for experimental testing. The protocol described here offers an extremely fast method for characterizing key proteins of a novel pathogen and for the identification of compounds that could inhibit or accelerate spreading of the disease.
Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Binding Sites , COVID-19/metabolism , Computational Biology , Computer Simulation , Crystallography, X-Ray , Drug Design , Drug Discovery , Drug Evaluation, Preclinical , Drug Repositioning , Host Microbial Interactions/drug effects , Host Microbial Interactions/physiology , Humans , Ligands , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , Solvents , User-Computer Interface , COVID-19 Drug TreatmentABSTRACT
AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Carotid Artery Injuries/therapy , Cell Proliferation , Cytoskeletal Proteins/metabolism , Genetic Therapy , Membrane Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Neointima , Nerve Tissue Proteins/metabolism , Repressor Proteins/metabolism , YY1 Transcription Factor/metabolism , Amino Acid Motifs , Animals , Calmodulin-Binding Proteins/genetics , Carotid Artery Injuries/genetics , Carotid Artery Injuries/metabolism , Carotid Artery Injuries/pathology , Carotid Artery, Common/metabolism , Carotid Artery, Common/pathology , Cattle , Cells, Cultured , Cytoskeletal Proteins/genetics , Disease Models, Animal , Hyperplasia , Membrane Proteins/genetics , Muscle, Smooth, Vascular/injuries , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Nerve Tissue Proteins/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Rabbits , Rats , Repressor Proteins/genetics , Signal Transduction , YY1 Transcription Factor/geneticsABSTRACT
The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.