RESUMO
Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTßR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTßR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTßR using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTßR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.
RESUMO
The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75, and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus-cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC-SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection.
Assuntos
Aedes , Vírus da Dengue , Dengue , Animais , Humanos , Vírus da Dengue/fisiologia , Pupa , Mosquitos VetoresRESUMO
The p38MAPK-MK2 signaling axis functions as an initiator of inflammation. Targeting the p38MAPK-MK2 signaling axis represents a direct therapeutic intervention of inflammatory diseases. We described here a novel role of andrographolide (AG), a small-molecule ent-labdane natural compound, as an inhibitor of p38MAPK-MK2 axis via MK2 degradation. AG was found to bind to the activation loop of MK2, located at the interface of the p38MAPK-MK2 biomolecular complex. This interaction disrupted the complex formation and predisposed MK2 to proteasome-mediated degradation. We showed that AG induced MK2 degradation in a concentration- and time-dependent manner and exerted its anti-inflammatory effects by enhancing the mRNA-destabilizing activity of tristetraprolin, thereby inhibiting pro-inflammatory mediator production (e.g., TNF-α, MCP-1). Administration of AG via intratracheal (i.t.) route to mice induced MK2 downregulation in lung alveolar macrophages, but not lung tissues, and prevented macrophage activation. Our study also demonstrated that the anti-inflammatory effects achieved by AG via MK2 degradation were more durable and sustained than that achieved by the conventional MK2 kinase inhibitors (e.g., PF-3644022). Taken together, our findings illustrated a novel mode of action of AG by modulating the p38MAPK-MK2 signaling axis and would pave the way for the development of a novel class of anti-inflammatory agents targeting MK2 for degradation by harnessing the privileged scaffold of AG.
Assuntos
Diterpenos , Proteínas Serina-Treonina Quinases , Camundongos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Diterpenos/farmacologia , Diterpenos/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry-a technique initially developed in mammalian cells-to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3'UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3'UTR, whereas AeStaufen interacts with both 3'UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen's role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3'UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.
Assuntos
Aedes , Vírus da Dengue , Dengue , Regiões 3' não Traduzidas/genética , Aedes/genética , Animais , Proteínas de Transporte/genética , Vírus da Dengue/genética , Humanos , Mamíferos , Mosquitos Vetores/genética , RNA Guia de Cinetoplastídeos , Proteínas de Ligação a RNA/genética , SalivaRESUMO
Mosquito saliva proteins modulate the human immune and hemostatic systems and control mosquito-borne pathogenic infections. One mechanism through which mosquito proteins may influence host immunity and hemostasis is their interactions with key human receptor proteins that may act as receptors for or coordinate attacks against invading pathogens. Here, using pull-down assays and proteomics-based mass spectrometry, we identified 11 Ae. aegypti salivary gland proteins (SGPs) (e.g., apyrase, Ae. aegypti venom allergen-1 [AaVA-1], neutrophil stimulating protein 1 [NeSt1], and D7 proteins), that interact with one or more of five human receptor proteins (cluster of differentiation 4 [CD4], CD14, CD86, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN], and Toll-like receptor 4 [TLR4]). We focused on CD4- and DC-SIGN-interacting proteins and confirmed that CD4 directly interacts with AaVA-1, D7, and NeST1 recombinant proteins and that AaVA-1 showed a moderate interaction with DC-SIGN using ELISA. Bacteria responsive protein 1 (AgBR1), an Ae. aegypti saliva protein reported to enhance ZIKV infection in humans but that was not identified in our pull-down assay moderately interacts with CD4 in the ELISA assay. Functionally, we showed that AaVA-1 and NeST1 proteins promoted activation of CD4+ T cells. We propose the possible impact of these interactions and effects on mosquito-borne viral infections such as dengue, Zika, and chikungunya viruses. Overall, this study provides key insight into the vector-host (protein-protein) interaction network and suggests roles for these interactions in mosquito-borne viral infections.
Assuntos
Aedes , Proteínas e Peptídeos Salivares , Alérgenos , Animais , Apirase , Humanos , Molécula 3 de Adesão Intercelular/metabolismo , Mosquitos Vetores , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14' position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.
Assuntos
Aedes , Trombose , Aedes/genética , Sequência de Aminoácidos , Animais , Coagulação Sanguínea , Fator XIIa/metabolismo , Inibidores de Proteases , Inibidores da Tripsina/farmacologiaRESUMO
Tripartite motif (TRIM) E3 ligases target specific substrates, including viral proteins, for proteasomal degradation, and are thus essential regulators of the innate antiviral response. TRIM69 ubiquitinates the non-structural NS3 protein of Dengue virus for its degradation by the host machinery. This antiviral strategy abrogates the immunosuppression mediated by the NS2B-NS3 protease complex. To understand how this host-driven antiviral response against Dengue virus, we sought to define the mode of interaction between human TRIM69 and Dengue NS2B-NS3 and the subsequent polyubiquitination of the protease by the E3 ligase. We show that NS2B-NS3Δpro is sufficient as a substrate for ubiquitination by TRIM69 using ELISA and in vitro assays. Using hydrogen-deuterium exchange mass spectrometry (HDXMS), we mapped the interface of the interaction between TRIM69 and NS2B-NS3Δpro, and propose a rationale for the binding and subsequent ubiquitination process. Furthermore, through sequence analysis, we showed that the regions targeted by TRIM69 on the DENV-2 NS3 protease (NS3Δpro) are well conserved across DENV serotypes and other flaviviruses, including Zika virus, West Nile virus, and Japanese encephalitis virus. Our results show the direct interactions of TRIM69 with viral proteins, provide mechanistic insights at a molecular level, and highlight the functional relevance of TRIM69 interacting with the Dengue viral protein. Collectively, our findings suggest that TRIM69 may act as a pan-antiflaviviral restriction factor.
Assuntos
Vírus da Dengue , Serina Endopeptidases , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Proteínas não Estruturais Virais , Dengue , Vírus da Dengue/fisiologia , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Serina Endopeptidases/química , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , Proteínas não Estruturais Virais/químicaRESUMO
Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with µ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.
Assuntos
Aedes , Sequência de Aminoácidos , Animais , Fibrinolisina , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Tripsina , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.
Assuntos
Aedes/enzimologia , Aedes/virologia , Carboxipeptidase B/metabolismo , Vírus da Dengue , Dengue/transmissão , Interações entre Hospedeiro e Microrganismos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidase B/química , Carboxipeptidase B/genética , Domínio Catalítico , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/fisiologia , Controle de Infecções , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki = 14.7 nM) is marginally less than that of bovine CPA1 (Ki = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.
Assuntos
Aedes/enzimologia , Carboxipeptidase B/química , Carboxipeptidases A/química , Proteínas de Insetos/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Carboxipeptidase B/antagonistas & inibidores , Carboxipeptidase B/genética , Carboxipeptidase B/metabolismo , Carboxipeptidases A/antagonistas & inibidores , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Domínio Catalítico , Bovinos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
microRNAs (miRNAs), a class of small and endogenous molecules that control gene expression, are broadly involved in biological processes. Although a number of cofactors that assist or antagonize let-7 miRNA biogenesis are well-established, more auxiliary factors remain to be investigated. Here, we identified SYNCRIP (Synaptotagmin Binding Cytoplasmic RNA Interacting Protein) as a new player for let-7a miRNA. SYNCRIP interacts with pri-let-7a both in vivo and in vitro. Knockdown of SYNCRIP impairs, while overexpression of SYNCRIP promotes, the expression of let-7a miRNA. A broad miRNA profiling analysis revealed that silencing of SYNCRIP regulates the expression of a set of mature miRNAs positively or negatively. In addition, SYNCRIP is associated with microprocessor complex and promotes the processing of pri-let-7a. Strikingly, the terminal loop of pri-let-7a was shown to be the main contributor for its interaction with SYNCRIP. Functional studies demonstrated that the SYNCRIP RRM2-3 domain can promote the processing of pri-let-7a. Structure-based alignment of RRM2-3 with other RNA binding proteins identified the residues likely to participate in protein-RNA interactions. Taken together, these findings suggest the promising role that SYNCRIP plays in miRNA regulation, thus providing insights into the function of SYNCRIP in eukaryotic development.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Regulação da Expressão Gênica/genética , Humanos , MicroRNAs/química , Ribonucleosídeo Difosfato Redutase/genéticaRESUMO
Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif "31WCXXC35". We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the repression of Prx1-mediated H2O2 decomposition leading to increased ROS accumulation in infected host cells. Increased ROS in turn prevented nuclear translocation of NF-κB and inhibition of NF-κB target genes, leading to bacterial survival and enhanced replication inside host cells. Targeting Trxlp-mediated virulence promises to attenuate E. piscicida infection.
Assuntos
Proteínas de Bactérias/metabolismo , Edwardsiella/fisiologia , Peroxirredoxinas/metabolismo , Transdução de Sinais , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sobrevivência Celular , Edwardsiella/genética , Edwardsiella/patogenicidade , Células HEK293 , Homeostase , Humanos , Peróxido de Hidrogênio/metabolismo , Imunidade , Modelos Moleculares , Mutação , NF-kappa B/metabolismo , Oxirredução , Transporte Proteico , Alinhamento de SequênciaRESUMO
BACKGROUND: Skin desquamation is facilitated by serine proteases KLK5 and KLK7, which are tightly regulated by lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI itself is controlled through degraded by mesotrypsin. Here, we sought to determine whether LEKTI exonic mutations associated with atopic dermatitis (AD) affect the protease inhibitory activity of LEKTI or its susceptibility to mesotrypsin degradation. METHODS: The inhibitory activities of the LEKTI domain 4 (D4) and D6 WT and AD-associated mutants on the enzyme activities of KLK5 and KLK7 were compared using fluorogenic substrates. A keratinocyte cell culture system using HaCat cells was established to assess the role of D6 WT and D386N on triggering inflammation via the induction of thymic stromal lymphopoietin (TSLP). A degradation assay was used to assess the susceptibility of D4 and D6 mutants to mesotrypsin degradation. RESULTS: Enzymatic assays revealed that the D6 D386N mutation affected the inhibitory activity of LEKTI on KLK5 but not KLK7. Other exonic mutations on D6 (N368S, V395M, and E420K) and D4 (R267Q) did not alter LEKTI inhibition. The D386N mutation disrupted the role of D6 in suppressing TSLP induction by KLK5 in HaCat cells. Although WT D4 is more susceptible to mesotrypsin degradation than WT D6, the D4 R267Q mutant was more resistant to mesotrypsin degradation, whereas the D6 E420K mutant showed enhanced mesotrypsin-mediated degradation. CONCLUSION: Exonic mutations in D6, which previously have been associated with AD, may cause a disruption of inhibitory activity on KLK5 or enhance the degradation by mesotrypsin.
Assuntos
Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Éxons , Mutação , Proteólise , Inibidor de Serinopeptidase do Tipo Kazal 5/genética , Citocinas/metabolismo , Dermatite Atópica/patologia , Epiderme/metabolismo , Células HaCaT , Haplótipos , Humanos , Inflamação/metabolismo , Calicreínas/química , Calicreínas/metabolismo , Queratinócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Domínios Proteicos/genética , Inibidor de Serinopeptidase do Tipo Kazal 5/metabolismo , Transdução de Sinais/genética , Tripsina/metabolismoRESUMO
Dengue symptoms include alteration of blood coagulation and fibrinolysis, causing severe hemorrhage and death. Here, we demonstrate that higher concentration of plasmin, the human fibrinolytic factor, in blood meal enhances dengue virus (DENV) infection in mosquito midgut and dissemination in mosquitoes. We also show that mosquitoes express a plasmin-selective Kazal-type inhibitor (AaTI) in the midgut to inhibit plasmin proteolysis and revert the enhanced infection. Using bio-layer interferometry, we show that DENV, plasmin, and AaTI interact to form a tripartite complex. Eventually, plasmin increases midgut internalization of dextran molecules and this is reverted by AaTI. Our study demonstrates that (1) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glycocalyx and enhancing DENV internalization and (2) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.
RESUMO
The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The Tm of A29G and V336I showed an increase by 5⯰C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345â¯K, and the MD data at 345â¯K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour.
Assuntos
Biocatálise , Engenharia de Proteínas/métodos , Serina Proteases/metabolismo , Temperatura , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Pseudomonas aeruginosa/enzimologia , Serina Proteases/química , Serina Proteases/isolamento & purificaçãoRESUMO
Despite being initially identified in the blood filtrate, LEKTI is a 15-domain Kazal-type inhibitor mostly known in the regulation of skin desquamation. In the current study, screening of serine proteases in blood coagulation cascade showed that LEKTI domain 4 has inhibitory activity toward only FXIa, whereas LEKTI domain 6 inhibits both FXIa and FXaB (bovine FXa). Nuclear magnetic resonance structural and dynamic experiments plus molecular dynamics simulation revealed that LEKTI domain 4 has enhanced backbone flexibility at the reactive-site loop. A model of the LEKTI-protease complex revealed that FXaB has a narrower S4 pocket compared with FXIa and hence prefers only small side-chain residues at the P4 position, such as Ala in LEKTI domain 6. Mutational studies combined with a molecular complex model suggest that both a more flexible reactive-site loop and a bulky residue at the P4 position make LEKTI domain 4 a weaker but highly selective inhibitor of FXIa.
Assuntos
Fator XI/antagonistas & inibidores , Fator X/antagonistas & inibidores , Inibidor de Serinopeptidase do Tipo Kazal 5/química , Inibidor de Serinopeptidase do Tipo Kazal 5/metabolismo , Animais , Sítios de Ligação , Coagulação Sanguínea , Bovinos , Fator X/química , Fator XI/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Inibidor de Serinopeptidase do Tipo Kazal 5/genética , Especificidade por SubstratoRESUMO
Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the recombinant protein using Bio-layer interferometry.
RESUMO
Centromeric identity and chromosome segregation are determined by the precise centromeric targeting of CENP-A, the centromere-specific histone H3 variant. The significance of the amino-terminal domain (NTD) of CENP-A in this process remains unclear. Here, we assessed the functional significance of each residue within the NTD of CENP-A from Schizosaccharomyces pombe (SpCENP-A) and identified a proline-rich 'GRANT' (Genomic stability-Regulating site within CENP-A N-Terminus) motif that is important for CENP-A function. Through sequential mutagenesis, we show that GRANT proline residues are essential for coordinating SpCENP-A centromeric targeting. GRANT proline-15 (P15), in particular, undergoes cis-trans isomerization to regulate chromosome segregation fidelity, which appears to be carried out by two FK506-binding protein (FKBP) family prolyl cis-trans isomerases. Using proteomics analysis, we further identified the SpCENP-A-localizing chaperone Sim3 as a SpCENP-A NTD interacting protein that is dependent on GRANT proline residues. Ectopic expression of sim3+ complemented the chromosome segregation defect arising from the loss of these proline residues. Overall, cis-trans proline isomerization is a post-translational modification of the SpCENP-A NTD that confers precise propagation of centromeric integrity in fission yeast, presumably via targeting SpCENP-A to the centromere.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas Nucleares/metabolismo , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Motivos de Aminoácidos , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Cromossomos Fúngicos/química , Teste de Complementação Genética , Instabilidade Genômica , Isomerismo , Cinética , Proteínas Nucleares/genética , Prolina/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Heterochromatin silencing is critical for genomic integrity and cell survival. It is orchestrated by chromodomain (CD)-containing proteins that bind to methylated histone H3 lysine 9 (H3K9me), a hallmark of heterochromatin. Here, we show that phosphorylation of tyrosine 41 (H3Y41p)-a novel histone H3 modification-participates in the regulation of heterochromatin in fission yeast. We show that a loss-of-function mutant of H3Y41 can suppress heterochromatin de-silencing in the centromere and subtelomere repeat regions, suggesting a de-silencing role for H3Y41p on heterochromatin. Furthermore, we show both in vitro and in vivo that H3Y41p differentially regulates two CD-containing proteins without the change in the level of H3K9 methylation: it promotes the binding of Chp1 to histone H3 and the exclusion of Swi6. H3Y41p is preferentially enriched on centromeric heterochromatin during M- to early S phase, which coincides with the localization switch of Swi6/Chp1. The loss-of-function H3Y41 mutant could suppress the hypersensitivity of the RNAi mutants towards hydroxyurea (HU), which arrests replication in S phase. Overall, we describe H3Y41p as a novel histone modification that differentially regulates heterochromatin silencing in fission yeast via the binding of CD-containing proteins.
Assuntos
Centrômero/genética , Heterocromatina/genética , Histonas/genética , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Pontos de Checagem do Ciclo Celular/genética , Centrômero/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Mutação com Perda de Função , Metilação , Fosforilação , Fase S/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMO
Human dermcidin (DCD) is an antimicrobial peptide secreted constitutively by sweat glands. The anionic derivative, DCD-1L, comprises of the N-terminal 47 residues of DCD and one additional leucine residue. A previous NMR structure of DCD-1L in 50% TFE showed a partial helical conformation, and its crystal structure in the presence of Zn2+ outlined a hexameric linear α-helical bundle. Three different models to describe membrane insertion were proposed but no conclusion was drawn. In the current study, the NMR structure of DCD-1L in SDS micelles showed an "L-shaped" molecule with three fully formed α-helices connected by flexible turns. Formation of these helices in DCD-1L in the presence of POPG vesicles suggests that the acidic C-terminal region of DCD-1L can suppress the binding of DCD-1L to POPG vesicles at basic but not acidic pH. Mutation of charged residues on the N-terminal and C-terminal regions of DCD-1L cause differences in POPG binding, suggesting distinct functional roles for these two regions. Charged residues from these two regions are also found to differentially affect Zn2+ coordination and aggregation of DCD-1L in the absence or presence of SDS, as monitored by 1D NMR. Our data agrees with one of the three models proposed.