RESUMEN
The aggregation of α-Synuclein (α-Syn) into amyloid fibrils is the hallmark of Parkinson's disease. Under stress or other pathological conditions, the accumulation of α-Syn oligomers is the main contributor to the cytotoxicity. A potential approach for treating Parkinson's disease involves preventing the accumulation of these α-Syn oligomers. In this study, we present a novel mechanism involving a conserved group of disorderly proteins known as small EDRK-rich factor (SERF), which promotes the aggregation of α-Syn through a cophase separation process. Using diverse methods like confocal microscopy, fluorescence recovery after photobleaching assays, solution-state NMR spectroscopy, and Western blot, we determined that the N-terminal domain of SERF1a plays a role in the interactions that occur during cophase separation. Within these droplets, α-Syn undergoes a gradual transformation from solid condensates to amyloid fibrils, while SERF1a is excluded from the condensates and dissolves into the solution. Notably, in vivo experiments show that SERF1a cophase separation with α-Syn significantly reduces the deposition of α-Syn oligomers and decreases its cellular toxicity under stress. These findings suggest that SERF1a accelerates the conversion of α-Syn from highly toxic oligomers to less toxic fibrils through cophase separation, thereby mitigating the biological damage of α-Syn aggregation.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Amiloide/química , Enfermedad de Parkinson/metabolismo , Separación de Fases , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Factores de Transcripción , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Células HeLa , Electricidad EstáticaRESUMEN
Cu/Zn superoxide dismutase 1 (SOD1) has a high propensity to misfold and form abnormal aggregates when it is subjected to oxidative stress or carries mutations associated with amyotrophic lateral sclerosis. However, the transition from functional soluble SOD1 protein to aggregated SOD1 protein is not completely clear. Here, we propose that liquid-liquid phase separation (LLPS) represents a biophysical process that converts soluble SOD1 into aggregated SOD1. We determined that SOD1 undergoes LLPS in vitro and cells under oxidative stress. Abnormal oxidation of SOD1 induces maturation of droplets formed by LLPS, eventually leading to protein aggregation and fibrosis, and involves residues Cys111 and Trp32. Additionally, we found that pathological mutations in SOD1 associated with ALS alter the morphology and material state of the droplets and promote the transformation of SOD1 to solid-like oligomers which are toxic to nerve cells. Furthermore, the fibrous aggregates formed by both pathways have a concentration-dependent toxicity effect on nerve cells. Thus, these combined results strongly indicate that LLPS may play a major role in pathological SOD1 aggregation, contributing to pathogenesis in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Superóxido Dismutasa-1 , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Mutación , Pliegue de Proteína , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Transición de FaseRESUMEN
Biomacromolecular condensates formed via phase separation establish compartments for the enrichment of specific compositions, which is also used as a biological tool to enhance molecule condensation, thereby increasing the efficiency of biological processes. Proteolysis-targeting chimeras (PROTACs) have been developed as powerful tools for targeted protein degradation in cells, offering a promising approach for therapies for different diseases. Herein, we introduce an intrinsically disordered region in the PROTAC (denoted PSETAC), which led to the formation of droplets of target proteins in the cells and increased degradation efficiency compared with PROTAC without phase separation. Further, using a nucleus targeting intrinsically disordered domain, the PSETAC was able to target and degrade nuclear-located proteins. Finally, we demonstrated intracellular delivery of PSETAC using lipid nanoparticle-encapsulated mRNA (mRNA-LNP) for the degradation of the endogenous target protein. This study established the PSETAC mRNA-LNP method as a potentially translatable, safe therapeutic strategy for the development of clinical applications based on PROTAC.
Asunto(s)
Proteolisis , ARN Mensajero , Proteolisis/efectos de los fármacos , Humanos , ARN Mensajero/genética , Nanopartículas/química , Lípidos/química , Células HeLa , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Separación de Fases , LiposomasRESUMEN
Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Tratamiento Farmacológico de COVID-19 , Péptidos/química , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Antivirales/química , Sitios de Unión/efectos de los fármacos , COVID-19/genética , COVID-19/virología , Diseño de Fármacos , Humanos , Simulación de Dinámica Molecular , Péptidos/genética , Péptidos/uso terapéutico , Unión Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.
Asunto(s)
Dolor Crónico , omega-Conotoxinas , Ratones , Animales , Dolor Crónico/tratamiento farmacológico , Micelas , omega-Conotoxinas/farmacología , Péptidos/farmacología , Bloqueadores de los Canales de Calcio/farmacologíaRESUMEN
Misfolding of TATA-box binding protein-associated factor 15 (TAF15) may cause neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Some mutations of prion-like domain (PrLD) have been detected in patients with sporadic ALS, suggesting the importance of TAF15-PrLD in ALS pathogenesis. Herein, combining experiments and molecular dynamics (MD) simulations, we investigated the influences of several TAF15-PrLD mutations on the amyloid fibril formation of TAF15-PrLD-extracted peptide segments, and identified an essential ß-amyloid-forming segment from TAF15-PrLD. A pathogenic mutation T2 E71G resulted in significantly enhanced aggregation of the TAF15-PrLD segment T2 (Y56GQSQSGYSQSYGGYENQ73). In addition, the peptide T2 with a strong ß-amyloid-forming tendency was able to induce the liquid to solid phase transition of TAF15-PrLD protein. Further study identified the SGYS motif as a critical segment that promoted the formation of amyloid fibrils, which maintained a stable ß-sheet structure through intermolecular hydrogen bonds and π-π stacking interaction. This work provides a clue to elucidate the molecular pathogenic mechanism of TAF15-associated neurodegenerative diseases, and will direct drug development targeting TAF15.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Priones , Factores Asociados con la Proteína de Unión a TATA , Amiloide , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Proteínas Priónicas/química , Priones/química , Factores Asociados con la Proteína de Unión a TATA/genéticaRESUMEN
Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.
Asunto(s)
Incrustaciones Biológicas , Cuerpos Extraños , Proteínas Intrínsecamente Desordenadas , Humanos , Incrustaciones Biológicas/prevención & control , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition.
Asunto(s)
Receptores de Hialuranos , Lipoilación/fisiología , Microdominios de Membrana , Simulación de Dinámica Molecular , Fosfatidilinositol 4,5-Difosfato , Membrana Celular/química , Membrana Celular/metabolismo , Biología Computacional , Citoplasma/química , Citoplasma/metabolismo , Receptores de Hialuranos/química , Receptores de Hialuranos/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMEN
Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26-38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people's view on inhibitor discovery for the checkpoint pair.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Receptores Inmunológicos/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Sustitución de Aminoácidos , Evolución Biológica , Simulación por Computador , Descubrimiento de Drogas , Simulación del Acoplamiento MolecularRESUMEN
Biofilm poses a serious challenge for the treatment of bacterial infections, as it endows bacteria a pronounced resistance to traditional antibiotics. Antimicrobial peptides (AMPs) are considered potential substitutes for antibiotics. Combinational use of AMPs with other compounds to exert antibiofilm effects has been proved to be an effective means to reduce their toxicity and maximize their antimicrobial activity. However, the combination of various AMPs with different action mechanisms is rarely investigated. A newly designed lytic AMP ZXR-2.3 combined with antibiofilm peptide IDR-1018 or KT2 was tested for the antibiofilm effect on mature Streptococcus mutans biofilms. In general, the combination of ZXR-2.3 + IDR-1018 displayed synergistic effect on both biofilm eradication and bacterial killing, while ZXR-2.3 + KT2 showed no obvious synergism. The confocal images of preformed S. mutans biofilms confirmed the effective bactericidal activity of ZXR-2.3 + IDR-1018. A tube system was applied to investigate the biofilm infection under a flow of medium and SEM images indicated the biofilm disruption and bacterial killing effects of ZXR-2.3 + IDR-1018. Quantitative RT-PCR analysis showed that IDR-1018 induced dramatic changes in the mRNA expressions of the quorum sensing (QS) related genes comC, comD, vicR, and vicK of S. mutans in mature biofilms, whereas the other peptides and ciprofloxacin did not cause obvious changes in these genes. This might explain the better synergism of ZXR-2.3 and IDR-1018. The results of this study provide a potential application using the combination of different AMPs in the treatment of mature biofilm infection.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Streptococcus mutans/patogenicidadRESUMEN
Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid-liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.
Asunto(s)
Enfermedades Neurodegenerativas/genética , Agregación Patológica de Proteínas/genética , Proteína FUS de Unión a ARN/química , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Asparagina/química , Asparagina/genética , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/patología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Humanos , Enfermedades Neurodegenerativas/patología , Péptidos/química , Péptidos/genética , Priones/química , Priones/genética , Agregación Patológica de Proteínas/patología , Dominios Proteicos/genética , Proteína FUS de Unión a ARN/genéticaRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a signaling lipid, mediating membrane trafficking and recruitment of proteins to membranes. A key example is the PIP2-dependent regulation of the adhesion of L-selectin to the cytoskeleton adaptors of the N-terminal subdomain of ezrin-radixin-moesin (FERM). The molecular details of the mediating behavior of multivalent anionic PIP2 lipids in this process, however, remain unclear. Here, we use coarse-grained molecular dynamics simulation to explore the mechanistic details of PIP2 in the transformation, translocation, and association of the FERM/L-selectin complex. We compare membranes of different compositions and find that anionic phospholipids are necessary for both FERM and the cytoplasmic domain of L-selectin to absorb on the membrane surface. The subsequent formation of the FERM/L-selectin complex is strongly favored by the presence of PIP2, which clusters around both proteins and triggers a conformational transition in the cytoplasmic domain of L-selectin. We are able to quantify the effect of PIP2 on the association free energy of the complex by means of a potential of mean force. We conclude that PIP2 behaves as an adhesive agent to enhance the stability of the FERM/L-selectin complex and identify key residues involved. The molecular information revealed in this study highlights the specific role of membrane lipids such as PIP2 in protein translocation and potential signaling.
Asunto(s)
Dominios FERM , Selectina L/metabolismo , Simulación de Dinámica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Membrana Celular/metabolismo , Selectina L/química , Unión Proteica , Transporte de ProteínasRESUMEN
The plasma transmembrane (TM) glycoprotein CD36 is critically involved in many essential signaling processes, especially the binding/uptake of long-chain fatty acids and oxidized low-density lipoproteins. The association of CD36 potentially activates cytosolic protein tyrosine kinases that are thought to associate with the C-terminal cytoplasmic tail of CD36. To understand the mechanisms by which CD36 mediates ligand binding and signal transduction, we have characterized the homo-oligomeric interaction of CD36 TM domains in membrane environments and with molecular dynamics (MD) simulations. Analysis of pyrene- and coumarin-labeled TM1 peptides in SDS by FRET confirmed the homodimerization of the CD36 TM1 peptide. Homodimerization assays of CD36 TM domains with the TOXCAT technique showed that its first TM (TM1) domain, but not the second TM (TM2) domain, could homodimerize in a cell membrane. Small-residue, site-specific mutation scanning revealed that the CD36 TM1 dimerization is mediated by the conserved small residues Gly12, Gly16, Ala20, and Gly23 Furthermore, molecular dynamics (MD) simulation studies demonstrated that CD36 TM1 exhibited a switching dimerization with two right-handed packing modes driven by the 12GXXXGXXXA20 and 20AXXG23 motifs, and the mutational effect of G16I and G23I revealed these representative conformations of CD36 TM1. This packing switch pattern of CD36 TM1 homodimer was further examined and confirmed by FRET analysis of monobromobimane (mBBr)-labeled CD36 TM1 peptides. Overall, this work provides a structural basis for understanding the role of TM association in regulating signal transduction via CD36.
Asunto(s)
Antígenos CD36/química , Simulación de Dinámica Molecular , Multimerización de Proteína , Secuencias de Aminoácidos , Antígenos CD36/genética , Antígenos CD36/metabolismo , Humanos , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin-ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL-1 transmembrane domains (TMDs) can substitute for the C320-dependent covalent bond to mediate the dimerization of PSGL-1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL-1 dimerization. The results of TOXCAT assays and Martini coarse-grained molecular dynamics (CG MD) simulations demonstrated that PSGL-1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL-1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL-1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL-1 dimer. These findings provide insights to understand the transmembrane association of PSGL-1 that is an important structural basis for PSGL-1 preferentially binding to P-selectin to achieve its biochemical and biophysical functions.
Asunto(s)
Leucina Zippers , Glicoproteínas de Membrana/química , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Escherichia coli/genética , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Dominios ProteicosRESUMEN
Polymer/inorganic nanocomposites exhibit special properties due to highly intimate interactions between organic and inorganic phases and thus have been deployed for various applications. Among them, nanocomposites with monolayer polymer coverage on the inorganic surface demonstrate the highest efficiency for applications. However, the controllable synthesis of the polymer monolayer in mesopores of inorganic substrates remains a challenge. In this study, poly(acrylic acid)/γ-alumina nanocomposites (PAA/alumina) were synthesized via the in situ polymerization of acrylic acid impregnated in mesopores of alumina. By applying the preneutralization of monomers, the polymerization was found to be highly controllable in generating monolayer PAA coverage. The formation of monolayers was verified by thermogravimetry, semiquantitative Fourier transform infrared spectroscopy, N2 adsorption-desorption, and Pb(II) adsorption. Alternatively, the organic loadings of PAA/alumina composite samples could be controlled in the range of 0.2 to 1.0 equiv of monolayer, together with the linearly correlated metal ion adsorption capacity. As calculated by the complexation model, one Pb(II) is combined with two carboxylate groups of PAA. The formation of the monolayer polymer inside mesoporous oxide channels represents a method for the development of highly promising functional nanocomposites.
RESUMEN
Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (LysâLeu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Citotoxinas/farmacología , Péptidos/farmacología , Sustitución de Aminoácidos , Antineoplásicos/síntesis química , Antineoplásicos/química , Proteínas Reguladoras de la Apoptosis/síntesis química , Proteínas Reguladoras de la Apoptosis/química , Línea Celular Tumoral , Citotoxinas/síntesis química , Citotoxinas/química , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mitocondrias/efectos de los fármacos , Péptidos/síntesis química , Péptidos/química , Venenos de Escorpión/química , Suero , Relación Estructura-ActividadRESUMEN
Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self-associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362-1370. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/metabolismo , Simulación de Dinámica Molecular , Proteínas Recombinantes de Fusión/química , Factor de Células Madre/química , 1,2-Dipalmitoilfosfatidilcolina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Perros , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Humanos , Cinética , Membrana Dobles de Lípidos/química , Ratones , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Multimerización de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Especificidad por Sustrato , Porcinos , TermodinámicaRESUMEN
Many lytic peptides contain a heptad sequence with leucine or isoleucine residues at "a" and "d" positions. However, their roles in the peptide-induced cytolytic process remain unclear. We have recently reported an anticancer lytic peptide ZXR-2 (FKIGGFIKKLWRSLLA), which contains a shortened zipper-like sequence with Ile/Leu at "a" and "d" positions. To understand the roles of these Ile/Leu residues, a series of analogs were constructed by sequentially replacing the Ile or Leu residue with alanine (Ala). Significant reduction of the cytolytic activity was observed when the Ile (3rd and 7th) and Leu (10th and 14th) residues at the "a" and "d" positions were substituted, while the replacement of the separate Leu (15th) residue had less effect. Based on the quenching of the intrinsic fluorescence of the peptides and their induced surface pressure changes of lipid monolayer, it was conjectured that the peptide ZXR-2 might insert into cell membranes from the C-terminal and to a depth of the W11 position. Accordingly, I3, I7, and L10 residues which mainly exposed in aqueous solution were more responsible for the peptide self-association on cell membranes, while L14, together with L15, might help peptide insert and anchor to cell membranes. These results are significant to elucidate the crucial roles of such Ile/Leu residues at "a" and "d" positions in peptide-peptide and peptide-membrane interactions to exert the membrane disruption activity of lytic peptides. With further understanding about the structure-activity relationship of lytic peptides, it would be helpful for designing novel anticancer lytic peptides.
Asunto(s)
Antineoplásicos/farmacología , Isoleucina/química , Leucina/química , Péptidos/farmacología , 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , Alanina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antineoplásicos/síntesis química , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colesterol/química , Células HEK293 , Células HeLa , Humanos , L-Lactato Deshidrogenasa/metabolismo , Liposomas/química , Péptidos/síntesis química , Fosfatidilserinas/química , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Electricidad Estática , Relación Estructura-ActividadRESUMEN
The lipid raft microenvironment is implicated in the generation of the pathological amyloid-ß (Aß) species in amyloid precursor protein (APP) that is associated with neurodegenerative diseases. Evidence shows that APP forms a transmembrane homodimer with changeable structures as a function of the membrane compositions. However, the molecular responsibility of the dimerization and structural alteration for the amyloidogenic process in segregated membranes remains largely unclear. Here, we performed multiple coarse grained (CG) simulations to explore the behavioral preference of the transmembrane domain of APP (called C99) that is affected by the lipid raft microenvironment. The results showed that C99 was anchored at the boundary of the lipid raft relying on the conserved hydrophobic motif of V710xxA713xxxV717xxxV721. Moreover, the dimerization of C99 was greatly destabilized by the lipid raft, which led to a susceptible switching packing conformation. The molecular driving forces were derived from the combined regulation of the saturated lipids and cholesterols rather than from the simple binding competition of cholesterol in the C99 dimerization. The molecular details of the differential dimerization in the raft-forming and bulk fluid bilayer environments were compared, and the structural information was helpful for further understanding the enzymolysis responsiveness of APP.