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SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.
Assuntos
COVID-19/imunologia , Aptidão Genética , Evasão da Resposta Imune , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/virologia , Humanos , Mutação , Filogenia , SARS-CoV-2/química , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , VirulênciaRESUMO
B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9 Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.
Assuntos
Galinhas , Epitopos de Linfócito T , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Vírus da Influenza A Subtipo H9N2/imunologia , Animais , Epitopos de Linfócito T/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismoRESUMO
Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.
Assuntos
Glicosiltransferases , beta-Glucanas , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , beta-Glucanas/metabolismo , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/metabolismoRESUMO
PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.
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Isquemia Encefálica , Oxigênio , Camundongos , Animais , Oxigênio/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Glucose/metabolismo , Isquemia Encefálica/metabolismo , Glicólise , Neurônios/metabolismo , OxirreduçãoRESUMO
RNA binding protein RBM10 participates in various RNA metabolism, and its decreased expression or loss of function by mutation has been identified in many human cancers. However, how its dysregulation contributes to human cancer pathogenesis remains to be determined. Here, we found that RBM10 expression was decreased in breast tumors, and breast cancer patients with low RBM10 expression presented poorer survival rates. RBM10 depletion in breast cancer cells significantly promotes the cellular proliferation and migration. We further demonstrated that RBM10 forms a triple complex with YBX1 and phosphatase 1B (PPM1B), in which PPM1B serves as the phosphatase of YBX1. RBM10 knock-down markedly attenuated association between YBX1 and PPM1B, leading to elevated levels of YBX1 phosphorylation and its nuclear translocation. Furthermore, cancer cells with RBM10 depletion had a significantly accelerated tumor growth in nude mice. Importantly, these enhanced tumorigenic phenotypes can be reversed by overexpression of PPM1B. Our findings provide the mechanistic bases for functional loss of RBM10 in promoting tumorigenicity, and are potentially useful in the development of combined therapeutic strategies for cancer patients with defective RBM10.
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Neoplasias da Mama , Carcinogênese , Animais , Camundongos , Humanos , Feminino , Camundongos Nus , Carcinogênese/genética , Fosforilação , Proliferação de Células/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Monoéster Fosfórico Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismoRESUMO
Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ÏC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.
Assuntos
Ligação Competitiva , Proteínas Intrinsicamente Desordenadas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Cinética , Espectroscopia de Ressonância Magnética , Camundongos , Mutação/genética , Peptídeos/química , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismoRESUMO
A number of studies have confirmed that Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ)-transcriptional enhanced associate domain (TEAD) activity is the driver of cancer development. However, the role and mechanism of the YAP/TAZ-TEAD pathway in cervical intraepithelial neoplasia (CIN) remain to be clarified. Therefore, this study was designed to observe the effect of YAP/TAZ-TEAD activity on the development of CIN and provide new ideas for the diagnosis and treatment of CIN. Firstly, cervical tissues were collected from CIN patients in different stages [CIN grade 1 (CIN1) tissue, CIN grade 2/3 (CIN 2/3) and squamous cell carcinoma (SCC)] and healthy volunteers. Next, the expression levels of YAP, TAZ and TEAD in cervical tissues and cells were observed by immunohistochemistry, qRT-PCR and western blot. Besides, Z172 and Z183 cells were transfected with siRNA-YAP/TAZ (si-YAP/TAZ) and YAP/TAZ overexpression vector (YAP-5SA). Also, Z172 cells were co-transfected with YAP-5SA and si-TEAD2/4. Subsequently, the stemness characteristics, glycolysis level and malignant transformation of cells in each group were observed by sphere-formation assay, commercial kit, MTT, Transwell, scratch experiment, xenotransplantation and western blot.The expression of YAP, TAZ and TEAD increased significantly in cervical cancer tissue and cell line at the stage of CIN2/3 and SCC. When YAP/TAZ was knocked down, the stemness characteristics, glycolysis level and malignant transformation of cancer cells were notably inhibited; while activating YAP/TAZ exhibited a completely opposite result. In addition, activating YAP/TAZ and knocking down the TEAD expression at the same time significant weakened the effect of activated YAP/TAZ signal on precancerous cells and reduced inhibitory effect of knocking down TEAD alone. YAP/TAZ-TEAD signal activates the characteristics and Warburg effect of cancer stem cells, thereby promoting the malignant transformation of CIN.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transformação Celular Neoplásica , Células-Tronco Neoplásicas , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Proteínas de Sinalização YAP , Humanos , Feminino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/metabolismo , Animais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismo , Linhagem Celular Tumoral , Camundongos , Efeito Warburg em Oncologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proliferação de Células/genética , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologiaRESUMO
Peptide-binding motif (PBM) model, a hierarchical clustering of HLA class I based on their binding specificity, was developed to predict immunopeptidome divergence. The effect of PBM mismatches on outcomes is unknown in HLA-haploidentical haematopoietic cell transplantation with post-transplant cyclophosphamide (PTCy-haplo). We therefore conducted a retrospective study using national registry data in PTCy-haplo. Overall, 1352 patients were included in the study. PBM-A bidirectional mismatch was associated with an increased risk of overall mortality in multivariable analysis (hazard ratio, 1.26; 95% confidence interval, 1.06 to 1.50; p = 0.010). None of relapse, non-relapse mortality (NRM) and graft-versus-host disease showed significant differences according to PBM-A bidirectional mismatch status in the entire cohort. The impact of PBM-A bidirectional mismatch on overall survival (OS) was preserved within the HLA-A genotype bidirectional mismatch population, and their lower OS stemmed from higher relapse rate in this population. The worse OS due to high NRM with PBM-A bidirectional mismatch was prominent in lymphoid malignancies receiving reduced-intensity conditioning. The PBM model may predict outcomes more accurately than HLA genotype mismatches. In conclusion, this study demonstrated that the presence of PBM-A bidirectional mismatch elevated the risk of mortality of PTCy-haplo. Avoiding PBM-A bidirectional mismatch might achieve better outcomes in PTCy-haplo.
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Osteoporosis is a metabolic bone loss disorder usually accompanied by overactivated osteoclast formation and increased bone resorption. Transcriptional co-activator with PDZ-binding motif (TAZ) is an emerging potential target for the treatment of osteoporosis. Our previous research showed that TAZ overexpression inhibited osteoclast formation while TAZ silencing had the opposite effect. In addition, TAZ knockout in mouse osteoclasts induced osteoporosis in animal experiments. XMU-MP-1 (XMU) is a selective MST1/2 inhibitor that can theoretically activate TAZ; however, its effect on osteoporosis remains unknown. In this study, we found that XMU treatment significantly increased TAZ expression in osteoclasts and inhibited osteoclast formation in vitro; however, this inhibitory effect was eliminated after the deletion of TAZ. Furthermore, XMU treatment upregulated TAZ expression in osteoclasts and alleviated ovariectomy (OVX)-induced osteoporosis in bilateral OVX mouse models. These findings suggest that XMU can effectively activate TAZ and that pharmacological activation of TAZ may be a promising option for the treatment of osteoporosis.
Assuntos
Osteogênese , Osteoporose , Camundongos , Animais , Feminino , Humanos , Osso Esponjoso , Osteoporose/etiologia , Osteoporose/induzido quimicamente , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , OvariectomiaRESUMO
The majority of patients with advanced colorectal cancer have chemoresistance to oxaliplatin, and studies on oxaliplatin resistance are limited. Our research showed that RNA-binding motif single-stranded interacting protein 1 (RBMS1) caused ferroptosis resistance in tumor cells, leading to oxaliplatin resistance. We employed bioinformatics to evaluate publically accessible data sets and discovered that RBMS1 was significantly upregulated in oxaliplatin-resistant colorectal cancer cells, in tandem with ferroptosis suppression. In vivo and in vitro studies revealed that inhibiting RBMS1 expression caused ferroptosis in colorectal cancer cells, restoring tumor cell sensitivity to oxaliplatin. Mechanistically, this is due to RBMS1 inducing prion protein translation, resulting in ferroptosis resistance in tumor cells. Validation of clinical specimens revealed that RBMS1 is similarly linked to tumor development and a poor prognosis. Overall, RBMS1 is a potential therapeutic target with clinical translational potential, particularly for oxaliplatin chemoresistance in colorectal cancer.
Assuntos
Neoplasias Colorretais , Ferroptose , Humanos , Oxaliplatina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA , Proteínas Priônicas/metabolismoRESUMO
Understanding the evolutionary strategies of the SARS-CoV-2 omicron variant is crucial for comprehending the COVID-19 pandemic and preventing future coronavirus pandemics. In this study, we determined the crystal structures of the receptor-binding domains (RBDs) from currently circulating omicron subvariants XBB.1 and XBB.1.5 (also the emerging XBB.1.9.1), each complexed with human ACE2. We studied how individual RBD residues evolved structurally in omicron subvariants, specifically how they adapted to human ACE2. Our findings revealed that residues 493 and 496, which exhibited good human ACE2 adaptation in pre-omicron variants, evolved to poor adaptation in early omicron subvariants (but with good adaption to mouse ACE2) and then reverted to good adaptation in recent omicron subvariants. This result is consistent with the hypothesis that non-human animals facilitated the evolution of early omicron subvariants. Additionally, residue 486, which exhibited good human ACE2 adaptation in early omicron subvariants, evolved to poor adaptation in later omicron subvariants and then returned to good adaptation in recent omicron subvariants. This result is consistent with the hypothesis that immune evasion facilitated the evolution of later omicron subvariants. Thus, our study suggests that both non-human animals and immune evasion may have contributed to driving omicron evolution at different stages of the pandemic. IMPORTANCE The sudden emergence and continued evolution of the SARS-CoV-2 omicron variant have left many mysteries unanswered, such as the origin of early omicron subvariants and the factors driving omicron evolution. To address these questions, we studied the crystal structures of human ACE2-bound receptor-binding domains (RBDs) from omicron subvariants XBB.1 and XBB.1.5 (XBB.1.9.1). Our in-depth structural analysis sheds light on how specific RBD mutations adapt to either human or mouse ACE2 and suggests non-human animals and immune evasion may have influenced omicron evolution during different stages of the pandemic. These findings provide valuable insights into the mechanisms underlying omicron evolution, deepen our understanding of the COVID-19 pandemic, and have significant implications for preventing future coronavirus pandemics.
Assuntos
Evolução Molecular , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Camundongos , Enzima de Conversão de Angiotensina 2/genética , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.
Assuntos
Proteínas do Envelope de Coronavírus , SARS-CoV-2 , Humanos , COVID-19 , Lisossomos/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas do Envelope de Coronavírus/metabolismo , Motivos de Aminoácidos , Liberação de VírusRESUMO
Transcriptional Co-Activator with PDZ-Binding Motif (TAZ, also known as WWTR1) is a downstream effector of the Hippo pathway, involved in the regulation of organ regeneration and cell differentiation in processes such as development and regeneration. TAZ has been shown to play a tumor-promoting role in various cancers. Currently, many studies focus on the role of TAZ in the process of mitophagy. However, the molecular mechanism and biological function of TAZ in renal clear cell carcinoma (KIRC) are still unclear. Therefore, we systematically analyzed the mRNA expression profile and clinical data of KIRC in The Cancer Genome Atlas (TCGA) dataset. We found that TAZ expression was significantly upregulated in KIRC compared with normal kidney tissue and was closely associated with poor prognosis of patients. Combined with the joint analysis of 36 mitophagy genes, it was found that TAZ was significantly negatively correlated with the positive regulators of mitophagy. Finally, our results confirmed that high expression of TAZ in KIRC inhibits mitophagy and promotes KIRC cell proliferation. In conclusion, our findings reveal the important role of TAZ in KIRC and have the potential to be a new target for KIRC therapy.
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Carcinoma de Células Renais , Neoplasias Renais , Mitofagia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Proliferação de Células/genética , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Mitofagia/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genéticaRESUMO
The N-terminal region of troponin T (TnT) does not bind any protein of the contractile machinery and the role of its hypervariability remains uncertain. In this review we report the evidence of the interaction between TnT and AMP deaminase (AMPD), a regulated zinc enzyme localized on the myofibril. In periods of intense muscular activity, a decrease in the ATP/ADP ratio, together with a decrease in the tissue pH, is the stimulus for the activation of the enzyme that deaminating AMP to IMP and NH3 displaces the myokinase reaction towards the formation of ATP. In skeletal muscle subjected to strong tetanic contractions, a calpain-like proteolytic activity produces the removal in vivo of a 97-residue N-terminal fragment from the enzyme that becomes desensitized towards the inhibition by ATP, leading to an unrestrained production of NH3. When a 95-residue N-terminal fragment is removed from AMPD by trypsin, simulating in vitro the calpain action, rabbit fast TnT or its phosphorylated 50-residue N-terminal peptide binds AMPD restoring the inhibition by ATP. Taking in consideration that the N-terminus of TnT expressed in human as well as rabbit white muscle contains a zinc-binding motif, we suggest that TnT might mimic the regulatory action of the inhibitory N-terminal domain of AMPD due to the presence of a zinc ion connecting the N-terminal and C-terminal regions of the enzyme, indicating that the two proteins might physiologically associate to modulate muscle contraction and ammonia production in fast-twitching muscle under strenuous conditions.
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AMP Desaminase , Troponina T , Animais , Humanos , Coelhos , Trifosfato de Adenosina , Amônia , AMP Desaminase/química , AMP Desaminase/metabolismo , Calpaína/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Peptídeos , Proteínas , Troponina T/química , Zinco/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is the most lethal subtype of BC, with unfavorable treatment outcomes. Evidence suggests the engagement of lncRNA MCM3AP-AS1 in BC development. This study investigated the action of MCM3AP-AS1 in chemoresistance of TNBC cells. Drug-resistant TNBC cell lines SUM159PTR and MDA-MB-231R were constructed by exposure to increasing concentrations of doxorubicin/docetaxel (DOX/DXL). MCM3AP-AS1 and miR-524-5p expression levels were determined by RT-qPCR. RNA binding motif 39 (RBM39) level was measured using Western blot. Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. The targeted binding of miR-524-5p with MCM3AP-AS1 or RBM39 was predicted by ECORI database and validated by dual-luciferase assays. The gain-and-loss of function assays were conducted in cells to investigate the interactions among MCM3AP-AS1, miR-524-5p, and RBM39. TNBC xenograft mouse models were established through subcutaneous injection of MCM3AP-AS1-silencing MDA-MB-231R cells and intraperitoneally administrated with DOX/DXL to verify the role of MCM3AP-AS1 in vivo. MCM3AP-AS1 was upregulated in drug-resistant TNBC cells, and MCM3AP-AS1 silencing could sensitize drug-resistant TNBC cells to chemotherapeutic drugs by promoting apoptosis. MCM3AP-AS1 targeted miR-524-5p. After DOX/DXL treatment, miR-524-5p inhibition partially reversed the effect of MCM3AP-AS1 silencing on inhibiting chemoresistance and promoting apoptosis of drug-resistant TNBC cells. miR-524-5p targeted RBM39. Silencing MCM3AP-AS1 promoted apoptosis via the miR-524-5p/RBM39 axis, thereby enhancing chemosensitivity of drug-resistant TNBC cells. MCM3AP-AS1 knockdown upregulated miR-524-5p, downregulated RBM39, and restrained tumor development in vivo. MCM3AP-AS1 silencing potentiates apoptosis of drug-resistant TNBC cells by upregulating miR-524-5p and downregulating RBM39, thereby suppressing chemoresistance in TNBC.
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The current wound-healing collagen mimetic peptides (CMPs) have limitations such as poor membrane permeability and protease susceptibility. Herein, the solid-phase peptide synthesis of CMPs containing the integrin binding motif GFOGER is reported. The peptide sequences also consist of lipophilic moieties (adamantane and palmitic acid) for improved membrane permeability and different collagen-inducing tripeptides, namely, Thr-Thr-Lys (TTK), Gly-His-Lys (GHK), Gln-Pro-Arg (QPR), and Glu-Glu-Met (EEM). The synthesized peptides were successfully characterized and purified using liquid chromatography-mass spectrometry and preparative high-performance liquid chromatography techniques, respectively. The palmitic acid moiety increased the hydrophobic nature of the peptides, and they were retained longer on the stationary material of the reverse phase C-18 column. The three-dimensional parallel-strand helical structure of peptide DGD-GG-GFOGER-GG-TTK-palmitate was obtained using nuclear magnetic resonance spectroscopy and circular dichroism. The synthesized peptides have the desired helical structure, which can promote integrin binding.
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Colágeno Tipo I , Ácido Palmítico , Sequência de Aminoácidos , Peptídeos/química , Colágeno , IntegrinasRESUMO
BACKGROUND: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. METHODS: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. RESULTS: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. CONCLUSIONS: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.
Assuntos
Antibacterianos , Biofilmes , Testes de Sensibilidade Microbiana , Staphylococcus epidermidis , Biofilmes/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/fisiologia , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Hemólise/efeitos dos fármacos , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9â Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxobutyrate in the pre-decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases that can perform valuable carboligation reactions.
Assuntos
Tiamina Pirofosfato , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/química , Liases/metabolismo , Liases/química , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Especificidade por Substrato , Modelos MolecularesRESUMO
The Wsc1, Wsc2, and Wsc3 proteins are essential cell surface sensors that respond to cell wall perturbation by activating the cell wall integrity pathway (CWIP). We show here that in situ production of cholesterol (in place of ergosterol) induces hyper-phosphorylation of Slt2, the MAPK of the CWIP, and upregulates cell wall biosynthesis. Deletion of all three Wsc genes in K. phaffii reverts these phenotypes. In the cholesterol-producing strain, both Wsc1 and Wsc3 accumulate in the plasma membrane. Close inspection of the transmembrane domains of all three Wsc proteins predicted by AlphaFold2 revealed the presence of CRAC sterol-binding motifs. Experiments using a photoreactive cholesterol derivative indicate intimate interaction of this sterol with the Wsc transmembrane domain, and this apparent sterol binding was abrogated in Wsc mutants with substitutions in the CRAC motif. We also observed cholesterol interaction with CRAC-like motifs in the transmembrane domains of mammalian integrins, analogs of Wsc proteins. Our results suggest that proper signaling of the Wsc sensors requires highly specific binding of the native endogenous terminal sterol, ergosterol.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Esteróis/metabolismo , Colesterol/metabolismo , Ergosterol/metabolismoRESUMO
This article was initially published in the Journal of Structural Biology, instead of the Journal of Structural Biology: X, due to a publisher error. We regret the inconvenience. The link to the article published in Journal of Structural Biology: X is presented below: https://www.sciencedirect.com/science/article/pii/S2590152423000090. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.