Molecular insights into STAT1a protein in rohu (Labeo rohita): unveiling expression profiles, SRC homology domain recognition, and protein-protein interactions triggered by poly I: C.

Introduction: STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a). Methods: The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations. Results: The full-length CDS of Labeo rohita STAT1a (LrSTAT1a) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a. Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106. Discussion: This study provides molecular insights into the STAT1a protein in Rohu (Labeo rohita) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC.

Uncovering the BIN1-SH3 interactome underpinning centronuclear myopathy.

Truncation of the protein-protein interaction SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1, Amphiphysin 2) protein leads to centronuclear myopathy. Here, we assessed the impact of a set of naturally observed, previously uncharacterized BIN1 SH3 domain variants using conventional in vitro and cell-based assays monitoring the BIN1 interaction with dynamin 2 (DNM2) and identified potentially harmful ones that can be also tentatively connected to neuromuscular disorders. However, SH3 domains are typically promiscuous and it is expected that other, so far unknown partners of BIN1 exist besides DNM2, that also participate in the development of centronuclear myopathy. In order to shed light on these other relevant interaction partners and to get a holistic picture of the pathomechanism behind BIN1 SH3 domain variants, we used affinity interactomics. We identified hundreds of new BIN1 interaction partners proteome-wide, among which many appear to participate in cell division, suggesting a critical role of BIN1 in the regulation of mitosis. Finally, we show that the identified BIN1 mutations indeed cause proteome-wide affinity perturbation, signifying the importance of employing unbiased affinity interactomic approaches.

Use of phosphotyrosine-containing peptides to target SH2 domains: Antagonist peptides of the Crk/CrkL-p130Cas axis.

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.

Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase.

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three ß strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Folding and Binding Kinetics of the Tandem of SH2 Domains from SHP2.

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.

Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

The Functional Roles of the Src Homology 2 Domain-Containing Inositol 5-Phosphatases SHIP1 and SHIP2 in the Pathogenesis of Human Diseases.

The src homology 2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are two proteins involved in intracellular signaling pathways and have been linked to the pathogenesis of several diseases. Both protein paralogs are well known for their involvement in the formation of various kinds of cancer. SHIP1, which is expressed predominantly in hematopoietic cells, has been implicated as a tumor suppressor in leukemogenesis especially in myeloid leukemia, whereas SHIP2, which is expressed ubiquitously, has been implicated as an oncogene in a wider variety of cancer types and is suggested to be involved in the process of metastasis of carcinoma cells. However, there are numerous other diseases, such as inflammatory diseases as well as allergic responses, Alzheimer's disease, and stroke, in which SHIP1 can play a role. Moreover, SHIP2 overexpression was shown to correlate with opsismodysplasia and Alzheimer's disease, as well as metabolic diseases. The SHIP1-inhibitor 3-α-aminocholestane (3AC), and SHIP1-activators, such as AQX-435 and AQX-1125, and SHIP2-inhibitors, such as K161 and AS1949490, have been developed and partly tested in clinical trials, which indicates the importance of the SHIP-paralogs as possible targets in the therapy of those diseases. The aim of this article is to provide an overview of the current knowledge about the involvement of SHIP proteins in the pathogenesis of cancer and other human diseases and to create awareness that SHIP1 and SHIP2 are more than just tumor suppressors and oncogenes.

SH3Ps recruit auxilin-like vesicle uncoating factors for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is an essential process of cargo uptake operating in all eukaryotes. In animals and yeast, BAR-SH3 domain proteins, endophilins and amphiphysins, function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains the BAR-SH3 domain proteins SH3P1-SH3P3, but their role is poorly understood. Here, we identify SH3Ps as functional homologs of endophilin/amphiphysin. SH3P1-SH3P3 bind to discrete foci at the plasma membrane (PM), and SH3P2 recruits late to a subset of clathrin-coated pits. The SH3P2 PM recruitment pattern is nearly identical to its interactor, a putative uncoating factor, AUXILIN-LIKE1. Notably, SH3P1-SH3P3 are required for most of AUXILIN-LIKE1 recruitment to the PM. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors rather than the uncoating phosphatases, synaptojanins. SH3P1-SH3P3 act redundantly in overall CME with the plant-specific endocytic adaptor TPLATE complex but not due to an SH3 domain in its TASH3 subunit.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Development of FRET Biosensor to Characterize CSK Subcellular Regulation.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

The molecular basis of Abelson kinase regulation by its αI-helix.

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2, and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal αI-helix exerts an additional force toward the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The αI-helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abl's activity by fixating the αI-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the αI-helix onto the KD/SH2SH3 interface.

In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function.

ABSTRACT: Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival, and its inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has, for more than 2 decades, generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using nonlymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas-mediated knockin mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and the morphology of lymphoid organs, as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control and mounted similar humoral immune responses as Y223F and wild-type animals. Collectively, our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase Cγ2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this posttranslational modification is dispensable for the function of BTK.

Prediction of virus-host interactions and identification of hot spot residues of DENV-2 and SH3 domain interactions.

Dengue virus, particularly serotype 2 (DENV-2), poses a significant global health threat, and understanding the molecular basis of its interactions with host cell proteins is imperative for developing targeted therapeutic strategies. This study elucidated the interactions between proline-enriched motifs and Src homology 3 (SH3) domain. The SH3 domain is pivotal in mediating protein-protein interactions, particularly by recognizing and binding to proline-rich regions in partner proteins. Through a computational pipeline, we analyzed the interactions and binding modes of proline-enriched motifs with SH3 domains, identified new potential DENV-2 interactions with the SH3 domain, and revealed potential hot spot residues, underscoring their significance in the viral life cycle. This comprehensive analysis provides crucial insights into the molecular basis of DENV-2 infection, highlighting conserved and serotype-specific interactions. The identified hot spot residues offer potential targets for therapeutic intervention, laying the foundation for developing antiviral strategies against Dengue virus infection. These findings contribute to the broader understanding of viral-host interactions and provide a roadmap for future research on Dengue virus pathogenesis and treatment.

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85ß/AP2-mediated endocytosis.

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable these multifaceted roles, the catalytic subunit p110 utilizes the multi-domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, its product PI(3,4,5)P3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and their relationship to PI(3,4,5)P3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains AP2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and increase both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.

p24-Tango1 interactions ensure ER-Golgi interface stability and efficient transport.

The eukaryotic p24 family, consisting of α-, ß-, γ- and δ-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion and that their localizations between ER exit site (ERES) and Golgi are interdependent in an α→ßδ→γ sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface.

Role of two modules controlling the interaction between SKAP1 and SRC kinases comparison with SKAP2 architecture and consequences for evolution.

SRC kinase associated phosphoprotein 1 (SKAP1), an adaptor for protein assembly, plays an important role in the immune system such as stabilizing immune synapses. Understanding how these functions are controlled at the level of the protein-protein interactions is necessary to describe these processes and to develop therapeutics. Here, we dissected the SKAP1 modular organization to recognize SRC kinases and compared it to that of its paralog SRC kinase associated phosphoprotein 2 (SKAP2). Different conserved motifs common to either both proteins or specific to SKAP2 were found using this comparison. Two modules harboring different binding properties between SKAP1 and SKAP2 were identified: one composed of two conserved motifs located in the second interdomain interacting at least with the SH2 domain of SRC kinases and a second one composed of the DIM domain modulated by the SH3 domain and the activation of SRC kinases. This work suggests a convergent evolution of the binding properties of some SRC kinases interacting specifically with either SKAP1 or SKAP2.

The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain.

The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Adaptor protein HIP-55 promotes macrophage M1 polarization through promoting AP-1 complex activation.

Overwhelming macrophage M1 polarization induced by malfunction of the renin-angiotensin-aldosterone system (RAAS) initiates inflammatory responses, which play a crucial role in various cardiovascular diseases. However, the underlying regulatory mechanism remains elusive. Here, we identified adaptor protein HIP-55 as a critical regulator of macrophage M1 polarization. The expression of HIP-55 was upregulated in M1 macrophage induced by Ang II. Overexpression of HIP-55 significantly promoted Ang II-induced macrophage M1 polarization, whereas genetic deletion of HIP-55 inhibited the Ang II-induced macrophage M1 polarization. Mechanistically, HIP-55 facilitated activator protein-1 (AP-1) complex activation induced by Ang II via promoting ERK1/2 and JNK phosphorylation. Moreover, blocking AP-1 complex activation can attenuate the function of HIP-55 in macrophage polarization. Collectively, our results reveal the role of HIP-55 in macrophage polarization and provide potential therapeutic insights for cardiovascular diseases associated with RAAS dysfunction.