Investigation of Signaling in Primary Populations of Human Senescent T Cells.

Mitogen-activated protein kinases, a family of three stress-related kinases, the Erks and Jnks and p38s, are activated by three-layer transphosphorylation cascades and are important for the activation, differentiation, and effector functions of lymphocytes. Recent studies on the aged immune systems from both humans and mice have uncovered a different mode of MAPK signaling that is independent of canonical activation cascades and instead occurs through simultaneous self-phosphorylation reactions within the sestrin-MAPK activation complex (sMAC), an immune-inhibitory complex not previously observed. In this chapter, we discuss methodologies to study these pathways at the population and single cell level, which allows rejuvenating immune cell differentiation and fate.

Unveiling citicoline's mechanisms and clinical relevance in the treatment of neuroinflammatory disorders.

Citicoline, a compound produced naturally in small amounts in the human body, assumes a pivotal role in phosphatidylcholine synthesis, a dynamic constituent of membranes of neurons. Across diverse models of brain injury and neurodegeneration, citicoline has demonstrated its potential through neuroprotective and anti-inflammatory effects. This review aims to elucidate citicoline's anti-inflammatory mechanism and its clinical implications in conditions such as ischemic stroke, head trauma, glaucoma, and age-associated memory impairment. Citicoline's anti-inflammatory prowess is rooted in its ability to stabilize cellular membranes, thereby curbing the excessive release of glutamate-a pro-inflammatory neurotransmitter. Moreover, it actively diminishes free radicals and inflammatory cytokines productions, which could otherwise harm neurons and incite neuroinflammation. It also exhibits the potential to modulate microglia activity, the brain's resident immune cells, and hinder the activation of NF-κB, a transcription factor governing inflammatory genes. Clinical trials have subjected citicoline to rigorous scrutiny in patients grappling with acute ischemic stroke, head trauma, glaucoma, and age-related memory impairment. While findings from these trials are mixed, numerous studies suggest that citicoline could confer improvements in neurological function, disability reduction, expedited recovery, and cognitive decline prevention within these cohorts. Additionally, citicoline boasts a favorable safety profile and high tolerability. In summary, citicoline stands as a promising agent, wielding both neuroprotective and anti-inflammatory potential across a spectrum of neurological conditions. However, further research is imperative to delineate the optimal dosage, treatment duration, and underlying mechanisms. Moreover, identifying specific patient subgroups most likely to reap the benefits of citicoline as a new therapy remains a critical avenue for exploration.

Kinome state is predictive of cell viability in pancreatic cancer tumor and cancer-associated fibroblast cell lines.

Numerous aspects of cellular signaling are regulated by the kinome-the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC.

Effect of 1800 MHz radiofrequency field exposure on cytokine and signal transduction protein expression in differentiated THP-1 cells.

PURPOSE: To evaluate the effects of 1800 MHz continuous wave (CW) and global system for mobile communications (GSM) modulated radiofrequency electromagnetic field (RFEMF) exposures on signal transduction (ST) protein and cytokine expression in differentiated human-derived monocytic THP-1 cells. MATERIALS AND METHODS: THP-1 cells were differentiated into adherent macrophage-like cells using phorbol 12-myristate 13-acetate (PMA). Following differentiation, cells were exposed to 1800 MHz CW or GSM modulated RFEMF for 0.5, 4, or 24 h at a specific absorption rate (SAR) of 0 (sham) or 2.0 W/kg. Concurrent positive controls (lipopolysaccharide for cytokines; anisomycin for ST proteins) and negative controls were included in each experiment. The expression levels of cytokines (GM-CSF, IFN-γ, IL-1ß, IL-6, IL-10, TNF-α) from culture media and phosphorylated and total ST proteins (CREB, JNK, NF-κB, p38, ERK1/2, Akt, p70S6k, STAT3, STAT5) from cell lysates were assessed using Milliplex magnetic bead array panels. RESULTS: No consistent effect of RFEMF exposure was observed in differentiated THP-1 cells. A statistically significant effect of overall exposure condition was observed for IL-6 with GSM modulation (P = 0.042), but no difference between RFEMF and sham for any exposure condition remained following adjustment for multiple comparisons (P ≥ 0.128). No statistically significant effect of exposure condition was detected for any other cytokine evaluated with either of the RFEMF modulations (P ≥ 0.078). There were no statistically significant changes in expression levels for any of the ST proteins under any studied exposure condition (P ≥ 0.320). CONCLUSIONS: In this study, no evidence of changes were observed in differentiated human derived THP-1 cells following exposure of up to 24 h to 1800 MHz RFEMF at SARs of 0 and 2.0 W/kg on the expression of ST proteins or cytokines.

Imperative role of adaptor proteins in macrophage toll-like receptor signaling pathways.

Macrophages are integral part of the body's defense against pathogens and serve as vital regulators of inflammation. Adaptor molecules, featuring diverse domains, intricately orchestrate the recruitment and transmission of inflammatory responses through signaling cascades. Key domains involved in macrophage polarization include Toll-like receptors (TLRs), Src Homology2 (SH2) and other small domains, alongside receptor tyrosine kinases, crucial for pathway activation. This review aims to elucidate the enigmatic role of macrophage adaptor molecules in modulating macrophage activation, emphasizing their diverse roles and potential therapeutic and investigative avenues for further exploration.

In our manuscript, we explore the vital role of adaptor proteins regarding ways, our immune cells, specifically macrophages, detect and respond to threats. These proteins act as crucial messengers, helping macrophages recognize harmful invaders and initiate the body's defense mechanisms. Understanding this process not only sheds light on how our immune system works but also holds promise for developing new therapies to combat infections and inflammatory diseases. Our findings offer insight into the intricate world of immune response, potentially paving the way for improved treatments for a range of health conditions.

Role of post-translational modifications of Sp1 in cardiovascular diseases.

Specific protein 1 (Sp1) is pivotal in sustaining baseline transcription as well as modulating cell signaling pathways and transcription factors activity. Through interactions with various proteins, especially transcription factors, Sp1 controls the expression of target genes, influencing numerous biological processes. Numerous studies have confirmed Sp1's significant regulatory role in the pathogenesis of cardiovascular disorders. Post-translational modifications (PTMs) of Sp1, such as phosphorylation, ubiquitination, acetylation, glycosylation, SUMOylation, and S-sulfhydration, can enhance or modify its transcriptional activity and DNA-binding stability. These modifications also regulate Sp1 expression across different cell types. Sp1 is crucial in regulating non-coding gene expression and the activity of proteins in response to pathophysiological stimuli. Understanding Sp1 PTMs advances our knowledge of cell signaling pathways in controlling Sp1 stability during cardiovascular disease onset and progression. It also aids in identifying novel pharmaceutical targets and biomarkers essential for preventing and managing cardiovascular diseases.

Structure, function, and immunomodulation of the CD8 co-receptor.

Expressed on the surface of CD8+ T cells, the CD8 co-receptor is a key component of the T cells that contributes to antigen recognition, immune cell maturation, and immune cell signaling. While CD8 is widely recognized as a co-stimulatory molecule for conventional CD8+ αß T cells, recent reports highlight its multifaceted role in both adaptive and innate immune responses. In this review, we discuss the utility of CD8 in relation to its immunomodulatory properties. We outline the unique structure and function of different CD8 domains (ectodomain, hinge, transmembrane, cytoplasmic tail) in the context of the distinct properties of CD8αα homodimers and CD8αß heterodimers. We discuss CD8 features commonly used to construct chimeric antigen receptors for immunotherapy. We describe the molecular interactions of CD8 with classical MHC-I, non-classical MHCs, and Lck partners involved in T cell signaling. Engineered and naturally occurring CD8 mutations that alter immune responses are discussed. The applications of anti-CD8 monoclonal antibodies (mABs) that target CD8 are summarized. Finally, we examine the unique structure and function of several CD8/mAB complexes. Collectively, these findings reveal the promising immunomodulatory properties of CD8 and CD8 binding partners, not only to uncover basic immune system function, but to advance efforts towards translational research for targeted immunotherapy.

Investigating the protective properties of Panax ginseng and its constituents against biotoxins and metal toxicity: a mechanistic review.

Natural toxins are toxic substances produced by living microorganisms and cause harmful effects to other creatures, but not the organisms themselves. Based on the sources, they are classified into fungal, microbial, herbal, algae, and animal biotoxins. Metals, the oldest toxicants, are not created or destroyed by human industry as elements, just concentrated in the biosphere. An antidote can counteract the toxic effects of a drug or toxin or mitigate the adverse effects of a harmful substance. The potential antidote effects of Panax ginseng in organ toxicity have been proved by many scientific research projects. Herein, we are going to gather a comprehensive mechanistic review of the antidotal effects of ginseng and its main constituents against natural toxins and metal toxicity. In this regard, a literate search has been done in PubMed/Medline, Science Direct, and Scopus from 2000 until 2024. The gathered data showed the protective impacts of this golden plant and its secondary metabolites against aflatoxin, deoxynivalenol, three-nitro propionic acid, ochratoxin A, lipopolysaccharide, nicotine, aconite, domoic acid, α-synuclein, amyloid ß, and glutamate as well as aluminum, cadmium, chrome, copper, iron, and lead. These antidotal effects occur by multi-functional mechanisms. It may be attributed to antioxidant, anti-inflammatory, and anti-apoptotic effects. Future research directions on the antidotal effects of ginseng against natural toxins and metal toxicity involve broadening the scope of studies to include a wider range of toxins and metals, exploring synergistic interactions with other natural compounds, and conducting more human clinical trials to validate the efficacy and safety of ginseng-based treatments.

Troxerutin Delays Skin Keratinocyte Senescence Induced by Ionizing Radiation Both In Vitro and In Vivo.

BACKGROUNDS: With the increasing demand for beauty and a healthy lifespan, studies regarding anti-skin aging have drawn much more attention than ever before. Skin cellular senescence, the primary cause of skin aging, is characterized by a cell cycle arrest in proliferating cells along with a senescence-associated secretory phenotype (SASP), which can be triggered by various internal or external stimuli. AIMS: Recent studies have made significant progress in the fields of anti-senescence and anti-aging. However, little is known about the roles and functions of natural compounds, particularly flavonoids, in skin cellular senescence studies. METHODS: In this study, using strategies including ionizing radiation (IR), senescence-associated ß galactosidase assay (SA-ß-Gal), immunofluorescence (IF), flow cytometry, PCR array, as well as in vivo experiments, we investigated the effects and roles of troxerutin (Trx), a natural flavonoid, in skin keratinocyte senescence. RESULTS: We found that Trx delays skin keratinocyte senescence induced by IR. Mechanistically, Trx protects the skin keratinocyte cells from senescence by alleviating reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and DNA damage caused by IR. In addition, Trx was also proved to relieve skin senescence and SASP secretion in vivo induced by IR stimulation. CONCLUSIONS: Altogether, our findings pointed to a new function of Trx in delaying stress-induced skin keratinocyte senescence, and should thus provide theoretical foundations for exploring novel strategies against skin aging.

Structure and distribution of sensor histidine kinases in the fungal kingdom.

Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.

SOCS6, an inhibitory factor in Japanese eel inhibits the type I IFN pathway and the MyD88-mediated NF-kB pathway.

SOCS family genes are a class of repressors in various signaling pathways of mammals involved in regulating immunity, growth, and development, but the information remains limited in teleost. The full-length cDNA sequence of the Japanese eel SOCS6 gene, named AjSOCS6, was first cloned and showed to encode 529 amino acids with a conserved SH2 structural domain and a typical structure of a C-terminal SOCS box. AjSOCS6 is evolutionarily close to that of rainbow trout and zebrafish. AjSOCS6 gene expression was observed across all tissues in Japanese eel, with the highest levels found in the intestine. In vivo studies showed that AjSOCS6 was significantly upregulated in the liver following exposure to LPS, poly I:C, and Aeromonas hydrophila infection. In vitro, stimulation with poly I:C, CpG, and A. hydrophila infection increased AjSOCS6 expression in Japanese eel liver cells. Subcellular localization revealed that AjSOCS6 was dispersed in the cytoplasm. Overexpressing AjSOCS6 significantly suppressed the expression of immune-related genes, such as c-Rel and p65 in the NF-κB pathway, IFN1, IFN2, and IFN4 in the type I IFN signaling pathway, and the downstream inflammatory factor IL-6 in Japanese eel liver cells. Conversely, knocking down AjSOCS6 in vitro in liver cells and in vivo in the liver, spleen, and kidney significantly upregulated these gene expressions. Co-transfection of AjSOCS6 with AjMyD88 into HEK293 cells significantly reduced NF-κB luciferase activities compared to AjMyD88 single-transfection groups, in a natural state and under LPS stimulation. These findings suggest that AjSOCS6 negatively regulates MyD88-dependent NF-κB and type I IFN signaling pathways, underscoring its role in the immune defense of fish against viral and bacterial infections.

Met1-linked ubiquitination in cell signaling regulation.

Met1-linked ubiquitination (Met1-Ub), also known as linear ubiquitination, is a newly identified atypical type of polyubiquitination that is assembled via the N-terminal methionine (Met1) rather than an internal lysine (Lys) residue of ubiquitin. The linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L and SHARPIN is the sole E3 ubiquitin ligase that specifically generates Met1-linked ubiquitin chains. The physiological role of LUBAC-mediated Met1-Ub has been first described as activating NF-κB signaling through the Met1-Ub modification of NEMO. However, accumulating evidence shows that Met1-Ub is broadly involved in other cellular pathways including MAPK, Wnt/ß-Catenin, PI3K/AKT and interferon signaling, and participates in various cellular processes including angiogenesis, protein quality control and autophagy, suggesting that Met1-Ub harbors a potent signaling capacity. Here, we review the formation and cellular functions of Met1-linked ubiquitin chains, with an emphasis on the recent advances in the cellular mechanisms by which Met1-Ub controls signaling transduction.

Copper Dyshomeostasis And Diabetic Complications: Chelation Strategies For Management.

Cuproptosis, An Emerging Concept In The Field Of Diabetes Research, Presents A Novel And Promising Perspective For The Effective Management Of Diabetes Mellitus And Its Associated Complications. Diabetes, Characterized By Chronic Hyperglycemia, Poses A Substantial Global Health Burden, With An Increasing Prevalence Worldwide. Despite Significant Progress In Our Understanding Of This Complex Metabolic Disorder, Optimal Therapeutic Strategies Still Remain Elusive. The Advent Of Cuproptosis, A Term Coined To Describe Copper-Induced Cellular Cell Death And Its Pivotal Role In Diabetes Pathogenesis, Opens New Avenues For Innovative Interventions. Copper, An Indispensable Trace Element, Plays A Pivotal Role In A Myriad Of Vital Biological Processes, Encompassing Energy Production, Bolstering Antioxidant Defenses, And Altered Cellular Signaling. However, In The Context Of Diabetes, This Copper Homeostasis Is Perturbed, Driven By A Combination Of Genetic Predisposition, Dietary Patterns, And Environmental Factors. Excessive Copper Levels Act As Catalysts For Oxidative Stress, Sparking Intricate Intracellular Signaling Cascades That Further Exacerbate Metabolic Dysfunction. In This Review, We Aim To Explore The Interrelationship Between Copper And Diabetes Comprehensively, Shedding Light On The Intricate Mechanisms Underpinning Cuproptosis. By Unraveling The Roles Of Copper Transporters, Copper-Dependent Enzymes, And Cuproptotic Signaling Pathways, We Seek To Elucidate Potential Therapeutic Strategies That Harness The Power Of Copper Modulation In Diabetes Management. This Insight Sets The Stage For A Targeted Approach To Challenge The Complex Hurdles Posed By Diabetes, Potentially Transforming Our Therapeutic Strategies In The Ongoing Fight Against This Pervasive Global Health Concern.

The Sinorhizobium meliloti nitrogen-fixing symbiosis requires CbrA-dependent regulation of a DivL and CckA phosphorelay.

The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For Sinorhizobium meliloti, cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of ΔcbrA were identified as alleles of divL and cckA. In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living ΔcbrA cells. Biochemical characterization of the S. meliloti hybrid histidine kinase CckA in vitro demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckAA373S suppressor protein of ΔcbrA has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity in vivo. These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of S. meliloti.IMPORTANCESinorhizobium meliloti is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important Medicago sativa. It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism.

Challenging the Norm: The Unrecognized Impact of Soluble Guanylyl Cyclase Subunits in Cancer.

Since the discovery of nitric oxide (NO), a long journey has led us to the present, during which much knowledge has been gained about its pathway members and their roles in physiological and various pathophysiological conditions. Soluble guanylyl cyclase (sGC), the main NO receptor composed of the sGCα1 and sGCß1 subunits, has been one of the central figures in this narrative. However, the sGCα1 and sGCß1 subunits remained obscured by the focus on sGC's enzymatic activity for many years. In this review, we restore the significance of the sGCα1 and sGCß1 subunits by compiling and analyzing available but previously overlooked information regarding their roles beyond enzymatic activity. We delve into the basics of sGC expression regulation, from its transcriptional regulation to its interaction with proteins, placing particular emphasis on evidence thus far demonstrating the actions of each sGC subunit in different tumor models. Exploring the roles of sGC subunits in cancer offers a valuable opportunity to enhance our understanding of tumor biology and discover new therapeutic avenues.

Impact of Metal Ions on Cellular Functions: A Focus on Mesenchymal Stem/Stromal Cell Differentiation.

Metals play a crucial role in the human body, especially as ions in metalloproteins. Essential metals, such as calcium, iron, and zinc are crucial for various physiological functions, but their interactions within biological networks are complex and not fully understood. Mesenchymal stem/stromal cells (MSCs) are essential for tissue regeneration due to their ability to differentiate into various cell types. This review article addresses the effects of physiological and unphysiological, but not directly toxic, metal ion concentrations, particularly concerning MSCs. Overloading or unbalancing of metal ion concentrations can significantly impair the function and differentiation capacity of MSCs. In addition, excessive or unbalanced metal ion concentrations can lead to oxidative stress, which can affect viability or inflammation. Data on the effects of metal ions on MSC differentiation are limited and often contradictory. Future research should, therefore, aim to clarify the mechanisms by which metal ions affect MSC differentiation, focusing on aspects such as metal ion interactions, ion concentrations, exposure duration, and other environmental conditions. Understanding these interactions could ultimately improve the design of biomaterials and implants to promote MSC-mediated tissue regeneration. It could also lead to the development of innovative therapeutic strategies in regenerative medicine.

Mathematical Modeling and Inference of Epidermal Growth Factor-Induced Mitogen-Activated Protein Kinase Cell Signaling Pathways.

The mitogen-activated protein kinase (MAPK) pathway is an important intracellular signaling cascade that plays a key role in various cellular processes. Understanding the regulatory mechanisms of this pathway is essential for developing effective interventions and targeted therapies for related diseases. Recent advances in single-cell proteomic technologies have provided unprecedented opportunities to investigate the heterogeneity and noise within complex, multi-signaling networks across diverse cells and cell types. Mathematical modeling has become a powerful interdisciplinary tool that bridges mathematics and experimental biology, providing valuable insights into these intricate cellular processes. In addition, statistical methods have been developed to infer pathway topologies and estimate unknown parameters within dynamic models. This review presents a comprehensive analysis of how mathematical modeling of the MAPK pathway deepens our understanding of its regulatory mechanisms, enhances the prediction of system behavior, and informs experimental research, with a particular focus on recent advances in modeling and inference using single-cell proteomic data.

The Raf/LIN-45 C-terminal distal tail segment negatively regulates signaling in Caenorhabditis elegans.

Raf protein kinases act as Ras-GTP sensing components of the ERK signal transduction pathway in animal cells, influencing cell proliferation, differentiation, and survival. In humans, somatic and germline mutations in the genes BRAF and RAF1 are associated with malignancies and developmental disorders. Recent studies shed light on the structure of activated Raf, a heterotetramer consisting of Raf and 14-3-3 dimers, and raised the possibility that a Raf C-terminal distal tail segment (DTS) regulates activation. We investigated the role of the DTS using the Caenorhabditis elegans Raf ortholog lin-45. Truncations removing the DTS strongly enhanced lin-45(S312A), a weak gain-of-function allele equivalent to RAF1 mutations found in patients with Noonan Syndrome. We genetically defined three elements of the LIN-45 DTS, which we termed the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. In the context of lin-45(S312A), mutation of each of these elements enhanced activity. We used AlphaFold to predict DTS protein interactions for LIN-45, fly Raf, and human BRAF, within the activated heterotetramer complex. We propose distinct functions for the LIN-45 DTS elements: i) the ASBS binds the kinase active site as an inhibitor, ii) phosphorylation of the KTP motif modulates DTS-kinase domain interaction, and iii) the aromatic cluster anchors the DTS in an inhibitory conformation. Human RASopathy-associated variants in BRAF affect residues of the DTS, consistent with these predictions. This work establishes that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans and provides a model for its function in other Raf proteins.

The Rac1-PAK1-Arp2/3 signaling axis regulates CHIKV nsP1-induced filopodia and optimal viral genome replication.

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex.IMPORTANCEThe alphavirus nsP1 protein dramatically remodels host cellular membranes, resulting in the formation of filopodia-like extensions. Although described decades ago, the molecular mechanisms controlling these membrane deformations and their functional importance remain elusive. Our study provides mechanistic insight, uncovering the critical role of the Rac1 GTPase, along with its downstream effectors PAK1 and the actin nucleator Arp2, in the nsP1-associated phenotype. Furthermore, we demonstrate that the Rac1-PAK1-Arp2 pathway is essential for optimal CHIKV genome replication. Our findings establish a parallel in the cellular mechanisms governing nsP1-induced plasma membrane reshaping and the production of a functional replication complex in infected cells.