GRB2: A dynamic adaptor protein orchestrating cellular signaling in health and disease.

GRB2, or Growth Factor Receptor-Bound Protein 2, is a pivotal adaptor protein in intracellular signal transduction pathways, particularly within receptor tyrosine kinase (RTK) signaling cascades. Its crystal structure reveals a modular architecture comprising a single Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains, facilitating dynamic interactions critical for cellular signaling. While SH2 domains recognize phosphorylated tyrosines, SH3 domains bind proline-rich sequences, enabling GRB2 to engage with various downstream effectors. Folding and binding studies of GRB2 in its full-length form and isolated domains highlight a complex interplay between its protein-protein interaction domains on the folding energy landscape and in driving its function. Being at the crosslink of many key molecular pathways in the cell, GRB2 possesses a role in cancer pathogenesis, particularly in mediating the Ras-mitogen activated protein kinase (MAPK) pathway. Thus, pharmacological targeting of GRB2 domains is a promising field in cancer therapy, with efforts focused on disrupting protein-protein interactions. However, the dynamic interplay driving GRB2 function suggests the presence of allosteric sites at the interface between domains that could be targeted to modulate the binding properties of its constituent domains. We propose that the analysis of GRB2 proteins from other species may provide additional insights to make the allosteric pharmacological targeting of GRB2 a more feasible strategy.

Pharmacological targeting of adaptor proteins in chronic inflammation.

BACKGROUND: Inflammation, a biological response of the immune system, can be triggered by various factors such as pathogens, damaged cells, and toxic compounds. These factors can lead to chronic inflammatory responses, potentially causing tissue damage or disease. Both infectious and non-infectious agents, as well as cell damage, activate inflammatory cells and trigger common inflammatory signalling pathways, including NF-κB, MAPK, and JAK-STAT pathways. These pathways are activated through adaptor proteins, which possess distinct protein binding domains that connect corresponding interacting molecules to facilitate downstream signalling. Adaptor molecules have gained widespread attention in recent years due to their key role in chronic inflammatory diseases. METHODS: In this review, we explore potential pharmacological agents that can be used to target adaptor molecules in chronic inflammatory responses. A comprehensive analysis of published studies was performed to obtain information on pharmacological agents. CONCLUSION: This review highlights the therapeutic strategies involving small molecule inhibitors, antisense oligonucleotide therapy, and traditional medicinal compounds that have been found to inhibit the inflammatory response and pro-inflammatory cytokine production. These strategies primarily block the protein-protein interactions in the inflammatory signaling cascade. Nevertheless, extensive preclinical studies and risk assessment methodologies are necessary to ensure their safety.

cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

Sequence and structural insights of monoleucine-based sorting motifs contained within the cytoplasmic domains of basolateral proteins.

Delivery to the correct membrane domain in polarized epithelial cells is a critical regulatory mechanism for transmembrane proteins. The trafficking of these proteins is directed by short amino acid sequences known as sorting motifs. In six basolaterally-localized proteins lacking the canonical tyrosine- and dileucine-based basolateral sorting motifs, a monoleucine-based sorting motif has been identified. This review will discuss these proteins with an identified monoleucine-based sorting motif, their conserved structural features, as well as the future directions of study for this non-canonical basolateral sorting motif.

Adaptor molecules mediate negative regulation of macrophage inflammatory pathways: a closer look.

Macrophages play a central role in initiating, maintaining, and terminating inflammation. For that, macrophages respond to various external stimuli in changing environments through signaling pathways that are tightly regulated and interconnected. This process involves, among others, autoregulatory loops that activate and deactivate macrophages through various cytokines, stimulants, and other chemical mediators. Adaptor proteins play an indispensable role in facilitating various inflammatory signals. These proteins are dynamic and flexible modulators of immune cell signaling and act as molecular bridges between cell surface receptors and intracellular effector molecules. They are involved in regulating physiological inflammation and also contribute significantly to the development of chronic inflammatory processes. This is at least partly due to their involvement in the activation and deactivation of macrophages, leading to changes in the macrophages' activation/phenotype. This review provides a comprehensive overview of the 20 adaptor molecules and proteins that act as negative regulators of inflammation in macrophages and effectively suppress inflammatory signaling pathways. We emphasize the functional role of adaptors in signal transduction in macrophages and their influence on the phenotypic transition of macrophages from pro-inflammatory M1-like states to anti-inflammatory M2-like phenotypes. This endeavor mainly aims at highlighting and orchestrating the intricate dynamics of adaptor molecules by elucidating the associated key roles along with respective domains and opening avenues for therapeutic and investigative purposes in clinical practice.

Driving autophagy - the role of molecular motors.

Most of the vesicular transport pathways inside the cell are facilitated by molecular motors that move along cytoskeletal networks. Autophagy is a well-explored catabolic pathway that is initiated by the formation of an isolation membrane known as the phagophore, which expands to form a double-membraned structure that captures its cargo and eventually moves towards the lysosomes for fusion. Molecular motors and cytoskeletal elements have been suggested to participate at different stages of the process as the autophagic vesicles move along cytoskeletal tracks. Dynein and kinesins govern autophagosome trafficking on microtubules through the sequential recruitment of their effector proteins, post-translational modifications and interactions with LC3-interacting regions (LIRs). In contrast, myosins are actin-based motors that participate in various stages of the autophagic flux, as well as in selective autophagy pathways. However, several outstanding questions remain with regard to how the dominance of a particular motor protein over another is controlled, and to the molecular mechanisms that underlie specific disease variants in motor proteins. In this Review, we aim to provide an overview of the role of molecular motors in autophagic flux, as well as highlight their dysregulation in diseases, such as neurodegenerative disorders and pathogenic infections, and ageing.

Evolution of the Major Components of Innate Immunity in Animals.

Innate immunity is present in all animals. In this review, we explore the main conserved mechanisms of recognition and innate immune responses among animals. In this sense, we discuss the receptors, critical for binding to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs); the downstream signaling proteins; and transcription factors that govern immune responses. We also highlight conserved inflammatory mediators that are induced after the recognition of DAMPs and PAMPs. At last, we discuss the mechanisms that are involved in the regulation and/or generation of reactive oxygen species (ROS), influencing immune responses, like heme-oxygenases (HOs).

Reconstitution of Organelle Transport Along Microtubules In Vitro.

In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.

Targeting Epsins to Inhibit Fibroblast Growth Factor Signaling While Potentiating Transforming Growth Factor-ß Signaling Constrains Endothelial-to-Mesenchymal Transition in Atherosclerosis.

BACKGROUND: Epsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODS: Using single-cell RNA sequencing combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knockout in Apoe-/- and lineage tracing/proprotein convertase subtilisin/kexin type 9 serine protease mutant viral-induced atherosclerotic mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTS: Single-cell RNA sequencing and lineage tracing revealed that epsins 1 and 2 promote EndoMT and that the loss of endothelial epsins inhibits EndoMT marker expression and transforming growth factor-ß signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in the Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 expression, which inhibits transforming growth factor-ß signaling and EndoMT. Epsins directly bind ubiquitinated fibroblast growth factor receptor-1 through their ubiquitin-interacting motif, which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic ubiquitin-interacting motif-containing peptide atheroma ubiquitin-interacting motif peptide inhibitor significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONS: We conclude that epsins potentiate EndoMT during atherogenesis by increasing transforming growth factor-ß signaling through fibroblast growth factor receptor-1 internalization and degradation. Inhibition of EndoMT by reducing epsin-fibroblast growth factor receptor-1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

Rabies virus P protein binds to TBK1 and interferes with the formation of innate immunity-related liquid condensates.

Viruses must overcome the interferon-mediated antiviral response to replicate and propagate into their host. Rabies virus (RABV) phosphoprotein P is known to inhibit interferon induction. Here, using a global mass spectrometry approach, we show that RABV P binds to TBK1, a kinase located at the crossroads of many interferon induction pathways, resulting in innate immunity inhibition. Mutations of TBK1 phosphorylation sites abolish P binding. Importantly, we demonstrate that upon RABV infection or detection of dsRNA by innate immunity sensors, TBK1 and its adaptor proteins NAP1 and SINTBAD form dynamic cytoplasmic condensates that have liquid properties. These condensates can form larger aggregates having ring-like structures in which NAP1 and TBK1 exhibit locally restricted movement. P binding to TBK1 interferes with the formation of these structures. This work demonstrates that proteins of the signaling pathway leading to interferon induction transiently form liquid organelles that can be targeted by viruses.

Identification of adaptor proteins by incorporating deep learning and PSSM profiles.

Adaptor proteins, also known as signal transduction adaptor proteins, are important proteins in signal transduction pathways, and play a role in connecting signal proteins for signal transduction between cells. Studies have shown that adaptor proteins are closely related to some diseases, such as tumors and diabetes. Therefore, it is very meaningful to construct a relevant model to accurately identify adaptor proteins. In recent years, many studies have used a position-specific scoring matrix (PSSM) and neural network methods to identify adaptor proteins. However, ordinary neural network models cannot correlate the contextual information in PSSM profiles well, so these studies usually process 20×N (N > 20) PSSM into 20×20 dimensions, which results in the loss of a large amount of protein information; This research proposes an efficient method that combines one-dimensional convolution (1-D CNN) and a bidirectional long short-term memory network (biLSTM) to identify adaptor proteins. The complete PSSM profiles are the input of the model, and the complete information of the protein is retained during the training process. We perform cross-validation during model training and test the performance of the model on an independent test set; in the data set with 1224 adaptor proteins and 11,078 non-adaptor proteins, five indicators including specificity, sensitivity, accuracy, area under the receiver operating characteristic curve (AUC) metric and Matthews correlation coefficient (MCC), were employed to evaluate model performance. On the independent test set, the specificity, sensitivity, accuracy and MCC were 0.817, 0.865, 0.823 and 0.465, respectively. Those results show that our method is better than the state-of-the art methods. This study is committed to improve the accuracy of adaptor protein identification, and laid a foundation for further research on diseases related to adaptor protein. This research provided a new idea for the application of deep learning related models in bioinformatics and computational biology.

Advances in molecular mechanisms of Metformin against aging / 中华老年医学杂志

Diabetes is a prevalent disease among the elderly population.Among the antidiabetic drugs, Metformin is the most commonly used, and has been found to have unique anti-aging effects through nutritional sensing regulation, maintenance of protein homeostasis, protection of mitochondrial function, alteration of intercellular communication, and telomere protection, among other mechanisms.The results of studies have shown that Metformin has significant anti-aging potential, thereby necessitating a thorough analysis and summary of its molecular mechanism and research progress in this regard.

Sorting of cargo in the tubular endosomal network.

Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor-ligand complex. Once inside, the cell then determines the fate of the receptor-ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes.

Targeting plant UBX proteins: AI-enhanced lessons from distant cousins.

Across all eukaryotic kingdoms, ubiquitin regulatory X (UBX) domain-containing adaptor proteins control the segregase cell division control protein 48 (CDC48), and thereby also control cellular proteostasis and adaptation. The structures and biological roles of UBX proteins in animals and fungi have garnered considerable attention. However, their counterparts in plants remain markedly understudied. Since 2021, the artificial intelligence (AI)-based algorithm AlphaFold has provided predictions of protein structural features that can be highly accurate. Predictions of the proteomes of all major model organisms are now freely accessible to the entire research community through user-friendly web interfaces. We propose that the combination of cross-kingdom comparison with AF analysis produces a wealth of testable hypotheses to inspire and guide experimental research on plant UBX domain-containing (PUX) proteins.

OtDUB from the Human Pathogen Orientia tsutsugamushi Modulates Host Membrane Trafficking by Multiple Mechanisms.

Host cell membrane-trafficking pathways are often manipulated by bacterial pathogens to gain cell entry, avoid immune responses, or to obtain nutrients. The 1,369-residue OtDUB protein from the obligate intracellular human pathogen Orientia tsutsugamushi bears a deubiquitylase (DUB) and additional domains. Here we show that OtDUB ectopic expression disrupts membrane trafficking through multiple mechanisms. OtDUB binds directly to the clathrin adaptor-protein (AP) complexes AP-1 and AP-2, and the OtDUB275-675 fragment is sufficient for binding to either complex. To assess the impact of OtDUB interactions with AP-1 and AP-2, we examined trans-Golgi trafficking and endocytosis, respectively. Endocytosis is reduced by two separate OtDUB fragments: one contains the AP-binding domain (OtDUB1-675), and the other does not (OtDUB675-1369). OtDUB1-675 disruption of endocytosis requires its ubiquitin-binding capabilities. OtDUB675-1369 also fragments trans- and cis-Golgi structures. Using a growth-based selection in yeast, we identified viable OtDUB675-1369 point mutants that also no longer caused Golgi defects in human cells. In parallel, we found OtDUB675-1369 binds directly to phosphatidylserine, and this lipid binding is lost in the same mutants. Together these results show that OtDUB contains multiple activities capable of modulating membrane trafficking. We discuss how these activities may contribute to Orientia infections.

In Vitro Reconstitution of Molecular Motor-Driven Mitochondrial Transport.

Intracellular trafficking of organelles driven by molecular motors underlies essential cellular processes. Mitochondria, the powerhouses of the cell, are one of the major cargoes of molecular motors. Efficient distribution of mitochondria ensures cellular fitness while defects in this process contribute to severe pathologies, such as neurodegenerative diseases. Reconstitution of the mitochondrial microtubule-based transport in vitro in a bottom-up approach provides a powerful tool to investigate the mitochondrial trafficking machinery in a controlled environment in the absence of complex intracellular interactions. In this chapter, we describe the procedures for achieving such reconstitution of mitochondrial transport.

14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic ß cells.

While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in ß cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in ß cell function, we generated ß cell-specific 14-3-3ζ-KO mice. Although no effects on ß cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the ß cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in ß cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan-14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of ß cell function and provides a deeper understanding of how insulin secretion is controlled in ß cells.

A case of neonatal-onset autoinflammation with infantile enterocolitis caused by a de novoNLRC4 gene variation / 中华围产医学杂志

This article reported a case of neonatal-onset autoinflammation with infantile enterocolitis (AIFEC) caused by NLRC4 gene mutation. The boy developed the disease in the neonatal period, presenting with recurrent fever, rash, hepatosplenomegaly and enterocolitis. Laboratory tests showed some indicators including ferritin and C-reactive protein were elevated. His condition was complicated by macrophage activation syndrome and anti-infective treatment was ineffective. High-throughput whole exome sequencing revealed a de novo heterozygous mutation of c.1021G>C (p.Val341Leu) in the NLRC4 gene and AIFEC was confirmed. AIFEC is a rare disease with no effective treatment at present, which can be developed in the neonatal period and diagnosed by whole exome sequencing.

Spatial regulation of microtubule-dependent transport by septin GTPases.

The intracellular long-range transport of membrane vesicles and organelles is mediated by microtubule motors (kinesins, dynein) which move cargo with spatiotemporal accuracy and efficiency. How motors navigate the microtubule network and coordinate their activity on membrane cargo are fundamental but poorly understood questions. New studies show that microtubule-dependent membrane traffic is spatially controlled by septins - a unique family of multimerizing GTPases that associate with microtubules and membrane organelles. We review how septins selectively regulate motor interactions with microtubules and membrane cargo. We posit that septins provide a novel traffic code that specifies the movement and directionality of select motor-cargo complexes on distinct microtubule tracks.