Conserved Signaling Pathway Research Articles

Ageing as “early-life inertia”: Disentangling life-history trade-offs along a lifetime of an individual

The theory that ageing evolves because of competitive resource allocation between the soma and the germline has been challenged by studies showing that somatic maintenance can be improved without impairing reproduction. However, it has been suggested that cost‐free improvement in somatic maintenance is possible only under a narrow range of benign conditions. Here, we show that experimental downregulation of insulin/IGF‐1 signaling (IIS) in C. elegans nematodes, a robustly reproducible life span‐ and health span‐extending treatment, reduces fitness in a complex variable environment when initiated during development but does not reduce fitness when initiated in adulthood. Thus, our results show that the costs and benefits of reduced IIS can be uncoupled when organisms inhabit variable environments, and, therefore, do not provide support for the resource allocation theory. Our findings support the theory that the force of natural selection on gene expression in evolutionarily conserved signaling pathways that shape life‐history traits declines after the onset of reproduction resulting in organismal senescence.

Regulation of Mitogen-Activated Protein Kinase Signaling Pathways by the Ubiquitin-Proteasome System and Its Pharmacological Potential.

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. SIGNIFICANCE STATEMENT: Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics.

Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye

SummaryNewts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L2 mutant and GMR-hid, GMR-GAL4 eye. L2 exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L2 and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.

Mechano-chemical feedback mediated competition for BMP signalling leads to pattern formation

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Targeting the Hippo pathway in heart repair.

The Hippo pathway is an evolutionarily and functionally conserved signalling pathway that controls organ size by regulating cell proliferation, apoptosis, and differentiation. Emerging evidence has shown that the Hippo pathway plays critical roles in cardiac development, homeostasis, disease, and regeneration. Targeting the Hippo pathway has tremendous potential as a therapeutic strategy for treating intractable cardiovascular diseases, such as heart failure. In this review, we summarize the function of the Hippo pathway in the heart. Particularly, we highlight the post-translational modification of Hippo pathway components, including the core kinases LATS1/2 and their downstream effectors YAP/TAZ, in different contexts, which has provided new insights and avenues in cardiac research.

Pak1 kinase controls cell shape through ribonucleoprotein granules.

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.

The Complex Genetic Basis and Multilayered Regulatory Control of Yeast Pseudohyphal Growth.

Eukaryotic cells are exquisitely responsive to external and internal cues, achieving precise control of seemingly diverse growth processes through a complex interplay of regulatory mechanisms. The budding yeast Saccharomyces cerevisiae provides a fascinating model of cell growth in its stress-responsive transition from planktonic single cells to a filamentous pseudohyphal growth form. During pseudohyphal growth, yeast cells undergo changes in morphology, polarity, and adhesion to form extended and invasive multicellular filaments. This pseudohyphal transition has been studied extensively as a model of conserved signaling pathways regulating cell growth and for its relevance in understanding the pathogenicity of the related opportunistic fungus Candida albicans, wherein filamentous growth is required for virulence. This review highlights the broad gene set enabling yeast pseudohyphal growth, signaling pathways that regulate this process, the role and regulation of proteins conferring cell adhesion, and interesting regulatory mechanisms enabling the pseudohyphal transition.

A platform utilizing Drosophila ovulation for nonhormonal contraceptive screening

A significant unmet need for new contraceptive options for both women and men remains due to side-effect profiles, medical concerns, and the inconvenience of many currently available contraceptive products. Unfortunately, the development of novel nonsteroidal female contraceptive medicine has been stalled in the last couple of decades due to the lack of effective screening platforms. Drosophila utilizes conserved signaling pathways for follicle rupture, a final step in ovulation that is essential for female reproduction. Therefore, we explored the potential to use Drosophila as a model to screen compounds that could inhibit follicle rupture and be nonsteroidal contraceptive candidates. Using our ex vivo follicle rupture assay, we screened 1,172 Food and Drug Administration (FDA)-approved drugs and identified six drugs that could inhibit Drosophila follicle rupture in a dose-dependent manner. In addition, we characterized the molecular actions of these drugs in the inhibition of adrenergic signaling and follicle rupture. Furthermore, we validated that three of the four drugs consistently inhibited mouse follicle rupture in vitro and that two of them did not affect progesterone production. Finally, we showed that chlorpromazine, one of the candidate drugs, can significantly inhibit mouse follicle rupture in vivo. Our work suggests that Drosophila ovulation is a valuable platform for identifying lead compounds for nonsteroidal contraceptive development and highlights the potential of these FDA-approved drugs as novel nonsteroidal contraceptive agents.

Probiotics Interactions and the Modulation of Major Signalling Pathways in Host Model Organism Caenorhabditis elegans.

Microorganisms live in the human digestive system and the gut microbiome constitutes part of our prime determining component for healthy aging and wellness. Gut microbiota has broad influences on its host, beginning from the digestion of food and nutrients absorption to protective roles against invading pathogens and host immune system regulation. Dysbiosis of the gut microbial composition has been linked to numerous diseases and there is a need to have a better grasp on what makes a 'good' gut microbiome. Caenorhabditiselegans(C.elegans) model organism is consideredas a well-suitedin-vivomodel system and, is at the frontline of probiotic research because of its well-defined characteristics and prolific nature. Most importantly,C.elegansfeeds on bacteria, which speeds up manipulations and investigations in probiotics research tremendously. With its unique salient features of short lifespan, and ease of propagation, different unknown probiotics biological roles can be measured at an organism level with precision in the form of worm's stress responses, survivability, and lifespan. In this review, new insights on the different mechanisms underlying the establishment of probiotics regulations of conserved signalling pathways such as p38 MAPK/SKN-1, DAF-2/DAF-16, and JNK-1/DAF-16 is highlighted based on information obtained from C. elegans studies. Along with the current state of knowledge and the uniqueness of C. elegans as a model organism, explorations of its future contribution and scope in synthetic biology and probiotics engineering strains are also addressed. This is expected to strengthen our understanding of probiotics roles and to facilitate novel discovery and applications, for specific therapeutics against age-related disorders and various pathophysiological conditions.

Asp56 in actin is critical for the full activity of the amino acid starvation-responsive kinase Gcn2.

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.

Canonical Notch Signaling Is Required for KrasG12D -Induced Myeloproliferative Neoplasm

Notch is a highly conserved signaling pathway that regulates cell-fate specification and tissue homeostasis in various contexts. Depending on the context, Notch signaling could be oncogenic or tumor suppressive. The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function (GOF) NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias.To investigate the role of Notch signaling in T cell acute lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN) induced by Ras mutation, we downregulated Notch signaling in oncogenic Kras (KrasG12D) mouse model, which develops T-ALL and MPN simultaneously. We took two independent genetic approaches to downregulate Notch signaling. Mx1-Cre-mediated conditional expression of dominant-negative Mastermind-like1 (DNMAML) potently inhibits canonical Notch-mediated transcriptional activation, while knocking out Protein O-fucosyltransferase 1 (Pofut1) reduces interactions of Notch receptors and their ligands and downregulates Notch signaling. To study the cell-autonomous functions of downregulating Notch signaling in oncogenic Kras -induced leukemogenesis, we transplanted either bone marrow cells or splenocytes into lethally irradiated mice.As expected, inhibition of Notch signaling significantly inhibited T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras, preserved intact Notch signaling, and acquired GOF Notch1 mutation(s). Surprisingly, we found that downregulation of Notch signaling inhibited MPN development in a cell-autonomous manner. Analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. RNA-Seq analysis of control and mutant myeloid progenitors (MPs) demonstrated that KrasG12D MPs exhibited enhanced oxidative phosphorylation and mitochondria respiration, and this aberrant gene expression pattern was largely restored by DNMAML expression. These metabolic changes were further validated in functional assays. Furthermore, we demonstrated that combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target Notch downstream mechanisms in oncogenic Ras-driven myeloid leukemias. DisclosuresPadron:Incyte: Honoraria, Research Funding.

Histidine Kinases: Diverse Functions in Plant Development and Responses to Environmental Conditions.

The two-component system (TCS), which is one of the most evolutionarily conserved signaling pathway systems, has been known to regulate multiple biological activities and environmental responses in plants. Significant progress has been made in characterizing the biological functions of the TCS components, including signal receptor histidine kinase (HK) proteins, signal transducer histidine-containing phosphotransfer proteins, and effector response regulator proteins. In this review, our scope is focused on the diverse structure, subcellular localization, and interactions of the HK proteins, as well as their signaling functions during development and environmental responses across different plant species. Based on data collected from scientific studies, knowledge about acting mechanisms and regulatory roles of HK proteins is presented. This comprehensive summary ofthe HK-related network provides a panorama of sophisticated modulating activities of HK members and gaps in understanding these activities, as well as the basis for developing biotechnological strategies to enhance the quality of crop plants.

Cryptochromes: Photochemical and structural insight into magnetoreception

Cryptochromes (CRYs) function as blue light photoreceptors in diverse physiological processes in nearly all kingdoms of life. Over the past several decades, they have emerged as the most likely candidates for light-dependent magnetoreception in animals, however, a long history of conflicts between in vitro photochemistry and in vivo behavioral data complicate validation of CRYs as a magnetosensor. In this review, we highlight the origins of conflicts regarding CRY photochemistry and signal transduction, and identify recent data that provides clarity on potential mechanisms of signal transduction in magnetoreception. The review primarily focuses on examining differences in photochemistry and signal transduction in plant and animal CRYs, and identifies potential modes of convergent evolution within these independent lineages that may identify conserved signaling pathways.

Two-component signaling system in plants: interaction network and specificity in response to stress and hormones.

Plants are exposed to various environmental challenges that can hamper their growth, development, and productivity. Being sedentary, plants cannot escape from these unfavorable environmental conditions and have evolved various signaling cascades to endure them. The two-component signaling (TCS) system is one such essential signaling circuitry present in plants regulating responses against multiple abiotic and biotic stresses. It is among the most ancient and evolutionary conserved signaling pathways in plants, which include membrane-bound histidine kinases (HKs), cytoplasmic histidine phosphotransfer proteins (Hpts), and nuclear or cytoplasmic response regulators (RRs). At the same time, TCS also involved in many signaling circuitries operative in plants in response to diverse hormones. These plant growth hormones play a significant role in diverse physiological and developmental processes, and their contribution to plant stress responses is coming up in a big way. Therefore, it is intriguing to know how TCS and various plant growth regulators, along with the key transcription factors, directly or indirectly control the responses of plants towards diverse stresses. The present review attempts to explore this relationship, hoping that this knowledge will contribute towards developing crop plants with enhanced climate resilience.

Whole Genome Level Analysis of the Wnt and DIX Gene Families in Mice and Their Coordination Relationship in Regulating Cardiac Hypertrophy.

The Wnt signaling pathway is an evolutionarily conserved signaling pathway that plays essential roles in embryonic development, organogenesis, and many other biological activities. Both Wnt proteins and DIX proteins are important components of Wnt signaling. Systematic studies of Wnt and DIX families at the genome-wide level may provide a comprehensive landscape to elucidate their functions and demonstrate their relationships, but they are currently lacking. In this report, we describe the correlations between mouse Wnt and DIX genes in family expansion, molecular evolution, and expression levels in cardiac hypertrophy at the genome-wide scale. We observed that both the Wnt and DIX families underwent more expansion than the overall average in the evolutionarily early stage. In addition, mirrortree analyses suggested that Wnt and DIX were co-evolved protein families. Collectively, these results would help to elucidate the evolutionary characters of Wnt and DIX families and demonstrate their correlations in mediating cardiac hypertrophy.

Multiomic analysis of stretched osteocytes reveals processes and signalling linked to bone regeneration and cancer

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine—regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

STRIPAK, a Key Regulator of Fungal Development, Operates as a Multifunctional Signaling Hub.

The striatin-interacting phosphatases and kinases (STRIPAK) multi subunit complex is a highly conserved signaling complex that controls diverse developmental processes in higher and lower eukaryotes. In this perspective article, we summarize how STRIPAK controls diverse developmental processes in euascomycetes, such as fruiting body formation, cell fusion, sexual and vegetative development, pathogenicity, symbiosis, as well as secondary metabolism. Recent structural investigations revealed information about the assembly and stoichiometry of the complex enabling it to act as a signaling hub. Multiple organellar targeting of STRIPAK subunits suggests how this complex connects several signaling transduction pathways involved in diverse cellular developmental processes. Furthermore, recent phosphoproteomic analysis shows that STRIPAK controls the dephosphorylation of subunits from several signaling complexes. We also refer to recent findings in yeast, where the STRIPAK homologue connects conserved signaling pathways, and based on this we suggest how so far non-characterized proteins may functions as receptors connecting mitophagy with the STRIPAK signaling complex. Such lines of investigation should contribute to the overall mechanistic understanding of how STRIPAK controls development in euascomycetes and beyond.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Getting Hedgehogs Where They Need to Go: Furin Cleavage Activates Dispatched for Sonic Hedgehog Transport

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is required for Hedgehog morphogen gradient formation and signaling to long-range targets. Molecular mechanisms that regulate Dispatched to control Hedgehog release are not yet defined. We reveal that Dispatched is activated by Furin-mediated cleavage at a conserved processing site in its first extracellular loop. Disruption of Furin cleavage alters Dispatched membrane trafficking and attenuates Sonic Hedgehog release from ligand-producing cells. CRISPR/Cas9 gene editing was used to generate transgenic mice harboring a cleavage site mutation in the endogenous Dispatched locus. Homozygous animals show neuronal patterning phenotypes and decreased viability, confirming a requirement for Dispatched processing in vivo. As such, Furin serves as a crucial post-translational regulator of Dispatched activity in Hedgehog-producing cells during tissue morphogenesis.

The Unfolded Protein Response and Autophagy on the Crossroads of Coronaviruses Infections.

The ability to sense and adequately respond to variable environmental conditions is central for cellular and organismal homeostasis. Eukaryotic cells are equipped with highly conserved stress-response mechanisms that support cellular function when homeostasis is compromised, promoting survival. Two such mechanisms – the unfolded protein response (UPR) and autophagy – are involved in the cellular response to perturbations in the endoplasmic reticulum, in calcium homeostasis, in cellular energy or redox status. Each of them operates through conserved signaling pathways to promote cellular adaptations that include re-programming transcription of genes and translation of new proteins and degradation of cellular components. In addition to their specific functions, it is becoming increasingly clear that these pathways intersect in many ways in different contexts of cellular stress. Viral infections are a major cause of cellular stress as many cellular functions are coopted to support viral replication. Both UPR and autophagy are induced upon infection with many different viruses with varying outcomes – in some instances controlling infection while in others supporting viral replication and infection. The role of UPR and autophagy in response to coronavirus infection has been a matter of debate in the last decade. It has been suggested that CoV exploit components of autophagy machinery and UPR to generate double-membrane vesicles where it establishes its replicative niche and to control the balance between cell death and survival during infection. Even though the molecular mechanisms are not fully elucidated, it is clear that UPR and autophagy are intimately associated during CoV infections. The current SARS-CoV-2 pandemic has brought renewed interest to this topic as several drugs known to modulate autophagy – including chloroquine, niclosamide, valinomycin, and spermine – were proposed as therapeutic options. Their efficacy is still debatable, highlighting the need to better understand the molecular interactions between CoV, UPR and autophagy.