RESUMO
Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network. Specifically, we show that O-GlcNAc modified HSP27 binds more strongly to the co-chaperone protein BAG3, which then promotes refolding of a model substrate by HSP70. We use proteomics to identify other potential HSP27 interactions that are changed by O-GlcNAc, including one that we confirm with another sHSP, αB-crystallin. These findings add additional evidence for O-GlcNAc as a switch for regulating protein-protein interactions and for modifications of chaperones as one mechanism by which O-GlcNAc protects against protein aggregation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Chaperonas Moleculares , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Redobramento de Proteína , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/química , Ligação Proteica , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Glicogênio , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Cristalografia por Raios X , Glicogênio/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Ligação ProteicaRESUMO
Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Relacionadas à Autofagia , Ubiquitinação , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Ubiquitina/metabolismo , Ubiquitina/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Ubiquitinadas/metabolismo , Proteínas Ubiquitinadas/isolamento & purificação , Proteínas Ubiquitinadas/química , Separação de FasesRESUMO
The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein-protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5's multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5's structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5's function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects.
Assuntos
Dendritos , Hipocampo , Proteínas com Domínio LIM , Domínios PDZ , Dendritos/metabolismo , Animais , Hipocampo/metabolismo , Hipocampo/citologia , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Domínios Proteicos , Neurônios/metabolismo , Ratos , Células Cultivadas , HumanosRESUMO
Sclerostin (SOST), a Wnt signaling pathway inhibitor, is involved in the pathogenesis of skeletal disorders. This study investigated the impact of the GKWWRPS motif on the PNAIG motif in Loop 2 of SOST, which is accountable for the interactions with the LRP6 protein that triggers the down-regulation of the Wnt signaling pathway. Single amino acid mutations on the GKWWRPS motif, hypothesized to have a probable stabilization effect towards the PNAIG motif, led to a significant reduction in the primary interactions between the SOST and LRP6 proteins. Protein-protein docking and molecular dynamic studies were conducted to investigate the role of the motif. The study found that a solitary mutation in the GKWWRPS motif significantly reduced the primary interactions between SOST and LRP6 proteins, except for probable cold-spot residues. The study's findings establish the GKWWRPS motif as a promising target for therapeutic interventions. Based on the obtained results, it can be inferred that alterations implemented within the GKWWRPS motif could lead to the destabilization of the PNAIG motif, which would directly modulate the interactions between the SOST and LRP6 proteins. The present investigation thus presents novel opportunities in the field of anti-sclerostin interventions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Mutação Puntual , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Simulação de Dinâmica Molecular , Simulação de Acoplamento MolecularRESUMO
Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Calorimetria , Proteínas Associadas aos Microtúbulos , Ligação Proteica , Termodinâmica , Calorimetria/métodos , Humanos , Ligantes , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Escherichia coli/metabolismo , Peptídeos/química , Peptídeos/metabolismoRESUMO
Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos , Proteínas de Ligação a DNA , Simulação de Dinâmica Molecular , Proteínas Musculares , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Humanos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dissulfetos/química , Dissulfetos/farmacologia , Ligação Proteica , Sítios de Ligação , Linhagem Celular Tumoral , Desenho Assistido por Computador , Desenho de FármacosRESUMO
The scaffold proteins JIP1 and JIP2 intervene in the c-Jun N-terminal kinase (JNK) pathway to mediate signaling specificity by coordinating the simultaneous assembly of multiple kinases. Using NMR, we demonstrate that JIP1 and JIP2 heterodimerize via their SH3 domains with the affinity of heterodimerization being comparable to homodimerization. We present the high-resolution crystal structure of the JIP2-SH3 homodimer and the JIP1-JIP2-SH3 heterodimeric complex. The JIP2-SH3 structure reveals how charge differences in residues at its dimer interface lead to formation of compensatory hydrogen bonds and salt bridges, distinguishing it from JIP1-SH3. In the JIP1-JIP2-SH3 complex, structural features of each homodimer are employed to stabilize the heterodimer. Building on these insights, we identify key residues crucial for stabilizing the dimer of both JIP1 and JIP2. Through targeted mutations in cellulo, we demonstrate a functional role for the dimerization of the JIP1 and JIP2 scaffold proteins in activation of the JNK signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Modelos Moleculares , Multimerização Proteica , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Cristalografia por Raios X , Ligação ProteicaRESUMO
Small ubiquitin-binding domains (UBDs) recognize small surface patches on ubiquitin with weak affinity, and it remains a conundrum how specific cellular responses may be achieved. Npl4-type zinc-finger (NZF) domains are â¼30 amino acid, compact UBDs that can provide two ubiquitin-binding interfaces, imposing linkage specificity to explain signaling outcomes. We here comprehensively characterize the linkage preference of human NZF domains. TAB2 prefers Lys6 and Lys63 linkages phosphorylated on Ser65, explaining why TAB2 recognizes depolarized mitochondria. Surprisingly, most NZF domains do not display chain linkage preference, despite conserved, secondary interaction surfaces. This suggests that some NZF domains may specifically bind ubiquitinated substrates by simultaneously recognizing substrate and an attached ubiquitin. We show biochemically and structurally that the NZF1 domain of the E3 ligase HOIPbinds preferentially to site-specifically ubiquitinated forms of NEMO and optineurin. Thus, despite their small size, UBDs may impose signaling specificity via multivalent interactions with ubiquitinated substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ligação Proteica , Ubiquitina , Humanos , Especificidade por Substrato , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Dedos de Zinco , Ubiquitinação , Quinase I-kappa B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Domínios Proteicos , Fosforilação , Células HEK293 , Proteínas de Membrana TransportadorasRESUMO
Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.
Assuntos
Chaperonas Moleculares , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Domínios ProteicosRESUMO
Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3-7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8-11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.
Assuntos
Citoesqueleto de Actina , Actinas , Forminas , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Microscopia Crioeletrônica , Forminas/química , Forminas/metabolismo , Forminas/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/ultraestrutura , Modelos Moleculares , Profilinas/química , Profilinas/metabolismo , Profilinas/ultraestrutura , Ligação ProteicaRESUMO
ErbB3-binding protein 1(Ebp1) has two isoforms, p42 Ebp1 and p48 Ebp1, both of which can regulate cell growth and differentiation. But these isoforms often have opposite effects, including contradictory roles in regulation of cell growth in different tissues and cells. P48 Ebp1 belongs to the full-length sequence, while conformational changes in the crystal structure of p42 Ebp1 reveals a lack of an α helix at the amino terminus. Due to the differences in the structures of these two isoforms, they have different binding partners and protein modifications. Ebp1 can function as both an oncogene and a tumor suppressor factor. However, the underlying mechanisms by which these two isoforms exert opposite functions are still not fully understood. In this review, we summarize the genes and the structures of protein of these two isoforms, protein modifications, binding partners and the association of different isoforms with diseases.
Assuntos
Isoformas de Proteínas , Humanos , Isoformas de Proteínas/metabolismo , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias/metabolismo , Ligação ProteicaRESUMO
BAG3 is a multifaceted protein characterised by having WW domain, PXXP motif and BAG domain. This protein gets upregulated during malignant transformation of cells and has been associated with poorer survival of patients. Procancerous activity of BAG domain of BAG3 is well documented. BAG domain interacts with ATPase domain of Hsp-70 preventing protein delivery to proteasome. This impediment results in enhanced cell survival, proliferation, resistance to apoptosis and chemoresistance. Besides BAG domain other two domains/motifs of BAG3 are under research vigilance to explore its further oncogenic role. This review summarises the role of different structural determinants of BAG3 in elevating oncogenesis. Based on the already existing findings, more interacting partners of BAG3 are anticipated. The anticipated partners of BAG3 can shed a wealth of information into the mechanistic insights of its proproliferative role. Proper insights into the mechanistic details adopted by BAG3 to curtail/elaborate activity of anticipated interacting partners can serve as a potent target for development of therapeutic interventions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Carcinogênese/metabolismo , Animais , Neoplasias/metabolismo , Neoplasias/patologia , Apoptose , Domínios ProteicosRESUMO
Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.
Assuntos
Proteína Substrato Associada a Crk , Peptídeos , Fosfotirosina , Proteínas Proto-Oncogênicas c-crk , Domínios de Homologia de src , Proteína Substrato Associada a Crk/metabolismo , Proteína Substrato Associada a Crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/química , Humanos , Fosfotirosina/metabolismo , Fosfotirosina/química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Ligação Proteica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Simulação de Acoplamento Molecular/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/químicaRESUMO
The SH2 domains of SHP2 play a crucial role in determining the function of the SHP2 protein. While the folding and binding properties of the isolated NSH2 and CSH2 domains have been extensively studied, there is limited information about the tandem SH2 domains. This study aims to elucidate the folding and binding kinetics of the NSH2-CSH2 tandem domains of SHP2 through rapid kinetic experiments, complementing existing data on the isolated domains. The results indicate that while the domains generally fold and unfold independently, acidic pH conditions induce complex scenarios involving the formation of a misfolded intermediate. Furthermore, a comparison of the binding kinetics of isolated NSH2 and CSH2 domains with the NSH2-CSH2 tandem domains, using peptides that mimic specific portions of Gab2, suggests a dynamic interplay between NSH2 and CSH2 in binding Gab2 that modulate the microscopic association rate constant of the binding reaction. These findings, discussed in the context of previous research on the NSH2 and CSH2 domains, enhance our understanding of the function of the SH2 domain tandem of SHP2.
Assuntos
Ligação Proteica , Dobramento de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Concentração de Íons de Hidrogênio , Cinética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/químicaRESUMO
While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.
Assuntos
Oxirredutases do Álcool , Polimerização , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/química , Microscopia Crioeletrônica , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Ligação Proteica , Células HEK293 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/genéticaRESUMO
Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
Assuntos
Chaperonas Moleculares , Domínios Proteicos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Sítios de Ligação , Humanos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Modelos Moleculares , Interações Hidrofóbicas e HidrofílicasRESUMO
The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fatores de Troca do Nucleotídeo Guanina , Proteínas rac1 de Ligação ao GTP , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Ligação Proteica , Conformação Proteica , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/químicaRESUMO
The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a ß-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Proteínas Associadas aos Microtúbulos , Ligação Proteica , Acetilação , Humanos , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Cristalografia por Raios X , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas NuclearesRESUMO
SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor-binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent but integrin- and clathrin-dependent manner and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9, and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to the entry of SARS-CoV-2.