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Neutrophils are key mediators of immune responses and inflammation in the body. Various neutrophil functions including chemotaxis, the rapid, directed migration of neutrophils towards chemical signals, are controlled by the recognition of chemoattractants by a specialized subfamily of G-protein-coupled receptors (GPCRs) including the widely studied formyl-peptide receptor 1 (FPR1) in humans. Receptor internalization prevents excessive activation of downstream ligand-induced signaling pathways, which allows cells to tune their sensitivity and limit inflammatory responses. Moreover, mutants deficient in FPR1-endocytosis migrate longer distances compared to their wild-type counterparts, suggesting that receptor internalization might act as a termination signal for neutrophil chemotaxis. Although precise regulation of chemotactic receptor internalization is critical for proper migration and function of neutrophils, the mechanism of FPR1 internalization and the role of internalization in chemotactic responses remain unclear.Taking genetic and cell biological approaches, we investigated the roles of both classic and unknown endocytosis regulators. Although their roles had been unclear, we found that multiple GPCR kinases (GRKs) and both β-arrestin 1 and 2 are important for FPR1 internalization using a flow-cytometry based endocytosis assay (Chapter 2). Moreover, we performed two parallel genome-wide CRISPR/Cas9 knock-out screens for the regulators of FPR1 surface expression and internalization (Chapter 3). Our results suggested multiple endocytic routes that FPR1 may follow, and this complexity may help explain conflicting data in the field. This screen identified potential new regulators of FPR1 endocytosis, including multiple subunits of endosome-associated protein complexes, small GTPases involved in membrane trafficking, and polarity signaling components. Through our ongoing investigation of some of these hits, we aim to validate and fully understand their involvement in chemoattractant receptor internalization and neutrophil chemotaxis. Better understanding of the roles of these regulators in FPR1 endocytosis and neutrophil function will unravel how the regulation of FPR1 endocytosis contributes to receptor-linked signaling events. Moreover, in Chapter 4, I summarize our findings on how ligand-dependent phosphorylation of another chemoattractant receptor, the LTB4 receptor, regulate its internalization. In conclusion, our efforts to gain insight into the mechanisms and biological consequences of chemoattractant receptor internalization broaden the current knowledge of regulation of these receptors and neutrophil chemotaxis. Accumulating evidence highlights the significance of neutrophil activity in the development of various ailments including inflammatory, autoimmune, and cardiovascular diseases. Gaining mechanistic insight into neutrophil function can help us develop novel strategies to modulate cell state and behavior during migration to control excessive or prolonged inflammation and thus prevent tissue damage.

Heart failure is a highly prevalent and costly disease, accounting for the number one source of hospitalization and healthcare expenditures in the United States. This massive unmet medical need is further underscored by the fact that there have been no new classes of heart failure therapeutics developed in the past twenty years, highlighting the urgent need for novel biological insights to pave the groundwork for drug development. The majority of existing therapies are largely targeted at cell surface neurohormonal pathways, but fail to completely alter disease progression. Interdicting downstream signaling and gene regulatory pathways may provide an alternative and complementary therapeutic strategy. My dissertation explores the roles of two family of kinases, salt-inducible kinases (SIKs) and cyclin dependent kinases (CDKs), in the setting of heart failure pathogenesis. The data generated in this body of work implicate two salt-inducible kinase isoforms, SIK1 and SIK3, as positive and negative regulators of pathologic cardiac remodeling, respectively, that can be pharmacologically targeted with small molecule inhibitors. I also uncover a role for CDK7, 12 and 13 as positive regulators of pathologic transcription and demonstrate proof-of-concept targeting of these kinases as an effective strategy for attenuating maladaptive cardiac remodeling in heart failure. Collectively, these findings advance our fundamental understanding of mechanisms that underlie the pathogenesis of heart failure and establish novel therapeutic entry points that may be exploited for the development of drugs to treat human heart failure.

Lysosomes are the main catabolic organelles of a eukaryotic cell and are critical for maintenance of cellular homeostasis. Macromolecules, organelles captured via autophagy, and material taken up by endocytosis are degraded in lysosomes. Consequently, lysosomes concentrate, store, recycle, and distribute metabolites crucial for biosynthetic processes. The lysosome also functions as a platform for regulating and coordinating signaling pathways. In particular, a master regulator of cell growth and proliferation – the mechanistic target of rapamycin complex 1 (mTORC1), an evolutionarily conserved serine/threonine protein kinase – is activated at the surface of the lysosome when nutrients are plentiful (e.g., amino acids potently promote mTORC1 activity). Cholesterol, a key component of biomembranes, also stimulates mTORC1 recruitment and activation at the lysosome. Thus, by integrating its degradative and signaling roles, the lysosome serves as a hub for nutrient sensing.In diseases known as lysosomal storage disorders, pathogenic levels of a particular metabolite accumulate in the lysosome due to the loss of function of a human gene required for catabolism or transport of a substrate normally digested in the lysosome. In Neimann-Pick type C (NPC) disease, the lysosomal cholesterol exporter, NPC1, is inoperative, causing accumulation of cholesterol within lysosomes, resulting in disruption of lysosomal function, which is propagated to other organelles (e.g., mitochondria) also compromising their function. Ultimately, this damage leads to progressive neurodegeneration in patients. The accumulation of cholesterol caused by loss of NPC1 also chronically hyperactivates mTORC1, further interfering with the regulation of cellular homeostasis.To increase our understanding of the factors that cause lysosomal failure in NPC disease, I examined the compositional and functional alterations that occur in lysosomes lacking NPC1 activity. Likewise, I studied how NPC1 loss triggers aberrant mTORC1 signaling and how dysregulated mTORC1 contributes to organelle pathogenesis. I first used organelle immuno-isolation in conjunction with proteomic profiling to uncover a pronounced proteolytic impairment of NPC lysosomes that is compounded by depletion of luminal hydrolases and enhanced susceptibility tomembrane damage. I also tested a panel of NPC1 mutants in NPC1-deficient cells and demonstrated that, cholesterol transport by NPC1 is tightly linked to regulation of mTORC1 activity, indicating that lysosomal cholesterol accumulation is the primary underlying cause of mTORC1 hyperactivation in NPC disease. Next, I demonstrated that genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis and lysosomal membrane integrity without correcting cholesterol accumulation, implicating mTORC1 hyperactivity as a main driver of downstream pathogenesis, including the loss of mitochondrial quality control and function. In agreement with those conclusions, I showed that mTORC1 inhibition reverses lysosomal and mitochondrial dysfunction in a neuronal model of NPC, extending my findings to a disease-relevant cellular context. Thus, targeting the cholesterol-mTORC1 signaling pathway may represent a novel therapeutic avenue in NPC that could complement or replace current approaches.

Proteins perform diverse jobs around the cell, including transporting nutrients and transmitting signals across membranes. For a long time, the traditional understanding of proteins has been that a protein's structure determines its function. However, recent research shows that more than 40% of human proteins contain intrinsically disordered domains, or pieces of protein lacking a stable structure. These domains have been found in diverse biological systems, including associated with the cytoskeleton, immune receptor tails, and as linkers between structured domains. Given their abundance and variety, it has become critical to understand how disordered domains behave. Here we investigate how characteristics of disordered proteins --- including length, binding site location, long- and short- lived interactions with other domains --- influence their binding kinetics. Specifically, we consider three disordered proteins in different biological systems: (1) multiply phosphorylated T cell receptor cytoplasmic tails, (2) formins --- a family of actin polymerization mediators, and (3) PD-1 tethered T cell signaling inhibition. For the T cell receptor cytoplasmic tails and formins, we use a freely-jointed chain model to simulate interactions between multisite disordered domains (e.g. T Cell Receptor, formin) and a binding partner (e.g. kinase, profilin-actin) represented as an idealized spherical domain. In each case, we demonstrate that disorder naturally leads to site-specific binding rates in multisite domains. For T cell receptor tails, we also demonstrate how physical changes due to post-translational modifications can give rise to positive cooperativity, negative cooperativity, and ultrasensitivity. We next show how variation between formin homologues, such as dimerization state, length, and binding site locations, can lead to changes in actin polymerization rate. Lastly, we analyze surface plasmon resonance experiments of the phosphatase SHP-1 dephosphorylating its tether, PD-1. We couple worm-like chain models with differential equations to extract binding kinetics, catalytic rates, and molecular reach parameters from the data. We use this data to quantify how changes in the lengths of disordered domains influence the rate and reach of reactions. Together, these results demonstrate the diversity and complexity of roles disordered proteins can play in cellular processes.

Protein kinases are enzymes that transfer the γ-phosphate of ATP to acceptor residues, most often serine, threonine or tyrosine, in proteins. Phosphorylation by protein kinases are integral to the signaling processes that control virtually all aspects of cellular physiology and phosphorylation is the most abundant post-translational modification in signal transduction networks. Covalent attachment of a phosphate group to a protein can cause a conformational change, introduce a new epitope that enhances or prevents interaction with another protein, increase or decrease the rate of turnover, and/or affect subcellular localization. In these ways, phosphorylation-dependent events can influence cell metabolism, morphology, and differentiation. Target of rapamycin (TOR) complex 2 (TORC2) is an evolutionarily conserved multi-subunit protein kinase and indispensable regulator of plasma membrane homeostasis. Genetic studies in yeast have demonstrated that the sole essential downstream target and effector of TORC2 is the protein kinase Ypk1 (and its paralog Ypk2), whose mammalian ortholog is SGK1. In humans, mutations in the TORC2 signaling network have been implicated in inflammatory disorders, metabolic diseases, and multiple cancers. In this doctoral dissertation, I describe the studies I undertook to explore novel aspects of TORC2-Ypk1 signaling. Because a Rab5-specific guanine nucleotide exchange factor (GEF), Muk1, was identified in a prior global screen for candidate Ypk1 targets, I investigated the role of Rab5-type GTPases in the TORC2 signaling network. I confirmed first that Muk1 is a substrate of Ypk1 and demonstrated that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Second, and strikingly, I found that yeast lacking its two Rab5 GEFs (Muk1 and Vps9), or its three Rab5 paralogs (Vps21/Ypt51, Ypt52 and Ypt53), or overexpressing Msb3 (a Rab5-directed GTPase-activating protein), all exhibited pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Finally, I showed that Vps21 co-immunoprecipitates with TORC2, and furthermore, that purified GTP-bound Vps21 is able to stimulate in vitro the activity of TORC2 immuno-enriched from Rab5-deficient cells. My findings suggest that TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling

Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients., The work was supported by Cancer Research UK (CRUK) C33043/A24478 (to V.S.-M., E.C.-M., O.M., and L.K.); Barts Charity (to V.S.M., R.S., and S.L.G.); The Harry J. Lloyd Charitable Trust (to J.L.O. and V.S.-M); and Comunidad de Madrid (Atraccion de Talento-2019-T1/BMD-13642) (to J.L.O.).

Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria (Staphylococcus sp., Listeria monocytogenes, etc.) and Gram-negative bacteria (Chlamydia sp., Salmonella sp., E. coli, etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins’ landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids’ more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies.

Nuclear receptors are classically defined as ligand-activated transcription factors that regulate key functions in reproduction, development, and physiology. Humans have 48 nuclear receptors, which when dysregulated are often linked to diseases. Because most nuclear receptors can be selectively activated or inactivated by small molecules, they are prominent therapeutic targets. The basic understanding of this family of transcription factors was accelerated in the 1980s upon the cloning of the first hormone receptors. During the next 20 years, a deep understanding of hormone signaling was achieved that has translated to numerous clinical applications, such as the development of standard-of-care endocrine therapies for hormonally driven breast and prostate cancers. A 2004 issue of this journal reviewed progress on elucidating the structures of nuclear receptors and their mechanisms of action. In the current issue, we focus on the broad application of new knowledge in this field for therapy across diverse disease states including cancer, cardiovascular disease, various inflammatory diseases, the aging brain, and COVID-19.

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H2O2) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H2O2, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H2O2, cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H2O2 via its enzymatic conversion to an amino acid-derived hydroperoxide that ‘escapes’ effective reductive inactivation to engage in selective oxidative activation of key target proteins., A major question in redox signaling is how H2O2 oxidizes target protein thiols in the presence of glutathione peroxidases and peroxiredoxins. We reveal signaling by H2O2 via its enzymatic conversion to an alkyl hydroperoxide that stereo-specifically escapes peroxidases and oxidizes target proteins.

Greater flamingos use cosmetic coloration by spreading uropygial secretions pigmented with carotenoids over their feathers, which makes the plumage redder. Because flamingos inhabit open environments that receive direct solar radiation during daytime, and carotenoids bleach when exposed to solar radiation, we expected that the plumage color would fade if there is no maintenance for cosmetic purposes. Here, we show that the concentrations of pigments inside feathers and on the surface of feathers were correlated, as well as that there was a correlation between the concentrations of pigments in the uropygial secretions and on the surface of feathers. There was fading in color (becoming less red) in feathers that received direct solar radiation when there was no plumage maintenance, but not so in others maintained in darkness. When we controlled for the initial color of feathers, the feathers of those individuals with higher concentration of pigments on the feather surfaces were those that lost less coloration after experimental exposure of feathers to sunny conditions. These results indicate that exposure to sunlight is correlated with the fading of feather color, which suggests that individuals need to regularly apply makeup to be more colorful. These results also reinforce the view that these birds use cosmetic coloration as a signal amplifier of plumage color. This may be important in species using highly variable habitats, such as wetlands, since the conditions experienced when molting may differ from those when the signal should be functional, usually months after molting., Greater flamingo plumage coloration fades when there is no frequent application of cosmetics.

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.

Summary Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs., Graphical abstract, Highlights • Superior NOTCH-mediated expansion of human HSPCs in hypoxic cultures • Culture under hypoxia mitigates ER stress in human HSPCs • Hypoxia potentiates NOTCH intracellular signaling in cultured human HSPCs, Larochelle and colleagues concurrently activate NOTCH and hypoxic pathways in ex vivo cultures of human adult HSPCs to enable a clinically relevant expansion of cells with long-term repopulating potential after transplantation. They characterize the molecular intersection between the two signaling pathways and demonstrate the important role of low oxygen tension in mitigating ER stress during NOTCH-mediated expansion of human HSPCs.

Alzheimer's Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Reportedly, two non-canonical Receptor Tyrosine Kinases (RTKs), ALK and RYK, both targets of the same micro RNA miR-1271, exhibit significant and consistent functional down-regulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS producing factor. Here we show that Grb2 and NOX4 play critical roles in reducing the severity of both the diseases. The study demonstrates that the abundance of Grb2 in degenerative conditions, in conjunction with NOX4, reverse cytoskeletal degradation by counterbalancing the network of small GTPases. PAX4, a transcription factor for both Grb2 and NOX4, emerges as the key link between the common pathways of AD and T2D. Down-regulation of both ALK and RYK through miR-1271, elevates the PAX4 level by reducing its suppressor ARX via Wnt/β-Catenin signaling. For the first time, this study brings together RTKs beyond Insulin Receptor (IR) family, transcription factor PAX4 and both AD and T2D pathologies on a common regulatory platform.

In response to increased anabolic demands to support their survival and growth, cancer cells rewire cellular signaling and metabolic networks. While analysis of this rewiring can reveal vulnerabilities within cancers, it is becoming clear that more profound understandings of fundamental cancer biology are required for the successful therapeutic exploitation of cancer signaling and metabolism, two very complex and highly-regulated systems with multiple pathways contributing to their plasticity and robustness.This thesis is the product of translational research efforts focused upon gaining a better understanding of fundamental aspects of cancer biology. The goals of the thesis are to 1) acquire novel insights of cancer biology to more precisely design therapeutic interventions to target cancers, more specifically pancreatic ductal adenocarcinoma (PDAC), one of most deadly cancers, and to 2) develop diagnostic tools that enable better characterization of metabolic characteristics of cancers.Chapter 1 concerns the development of a clinically-applicable positron emission tomography (PET) probe for monitoring nucleotide metabolism at the whole-body level in a non-invasive way. This study makes us think about biology across different scales, from molecular to whole-body levels.In Chapter 2, the synthetic lethality screening tethered to chloroquine, an inhibitor of lysosome-dependent recycling pathways, which PDAC cells rely on to generate metabolic substrates is performed. The findings reveal that the inhibition of lysosomal recycling makes PDAC cells rely on replication stress response pathway for the survival due to impaired de novo nucleotide biosynthesis.In chapter 3, I use multiple models of KRAS G12C mutant PDAC to profile the adaptive resistance mechanisms within cellular signaling and metabolic networks which arise from direct inhibition of mutant KRAS, the oncogenic driver of PDAC. This was followed by mapping the impacts of co-inhibition of oncogenic KRAS and the identified adaptive resistance mechanisms. Metabolomic analyses reveal metabolic vulnerabilities induced by co-inhibition of oncogenic KRAS and the adaptive resistance. In the final chapter, I summarize ongoing projects and future directions that concern fundamental biology of PDAC metabolism.

Plants interact with other organisms employing volatile organic compounds (VOCs). The largest group of plant-released VOCs are terpenes, comprised of isoprene, monoterpenes, and sesquiterpenes. Mono- and sesquiterpenes are well-known communication compounds in plant-insect interactions, whereas the smallest, most commonly emitted terpene, isoprene, is rather assigned a function in combating abiotic stresses. Recently, it has become evident that different volatile terpenes also act as plant-to-plant signaling cues. Upon being perceived, specific volatile terpenes can sensitize distinct signaling pathways in receiver plant cells, which in turn trigger plant innate immune responses. This vastly extends the range of action of volatile terpenes, which not only protect plants from various biotic and abiotic stresses, but also convey information about environmental constraints within and between plants. As a result, plant-insect and plant-pathogen interactions, which are believed to influence each other through phytohormone crosstalk, are likely equally sensitive to reciprocal regulation via volatile terpene cues. Here, we review the current knowledge of terpenes as volatile semiochemicals and discuss why and how volatile terpenes make good signaling cues. We discuss how volatile terpenes may be perceived by plants, what are possible downstream signaling events in receiver plants, and how responses to different terpene cues might interact to orchestrate the net plant response to multiple stresses. Finally, we discuss how the signal can be further transmitted to the community level leading to a mutually beneficial community-scale response or distinct signaling with near kin.

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.

The COVID-19 pandemic has prompted intense research efforts into elucidating mechanisms of coronavirus pathogenesis and to propose antiviral interventions. The interferon (IFN) response is the main antiviral component of human innate immunity and is actively suppressed by several non-structural SARS-CoV-2 proteins, allowing viral replication within human cells. Differences in IFN signalling efficiency and timing have emerged as central determinants of the variability of COVID-19 disease severity between patients, highlighting the need for an improved understanding of host–pathogen interactions that affect the IFN response. ADP-ribosylation is an underexplored post-translational modification catalyzed by ADP-ribosyl transferases collectively termed poly(ADP-ribose) polymerases (PARPs). Several human PARPs are induced by the IFN response and participate in antiviral defences by regulating IFN signalling itself, modulating host processes such as translation and protein trafficking, as well as directly modifying and inhibiting viral target proteins. SARS-CoV-2 and other viruses encode a macrodomain that hydrolyzes ADP-ribose modifications, thus counteracting antiviral PARP activity. This mini-review provides a brief overview of the known targets of IFN-induced ADP-ribosylation and the functions of viral macrodomains, highlighting several open questions in the field.

The twelfth annual workshop of the European Network for Breast Development and Cancer focused on methods in mammary gland biology and breast cancer, was scheduled to take place on March 26–28, 2020, in Weggis, Switzerland. Due to the COVID-19 pandemic, the meeting was rescheduled twice and eventually happened as a virtual meeting on April 22 and 23, 2021. The main topics of the meeting were branching and development of the mammary gland, tumor microenvironment, circulating tumor cells, tumor dormancy and breast cancer metastasis. Novel and unpublished findings related to these topics were presented, with a particular focus on the methods used to obtain them. Virtual poster sessions were a success, with many constructive and fruitful interactions between researchers and covered many areas of mammary gland biology and breast cancer.

The Hippo pathway is a dynamic cellular signalling nexus that regulates differentiation and controls cell proliferation and death. If the Hippo pathway is not precisely regulated, the functionality of the upstream kinase module is impaired, which increases nuclear localisation and activity of the central effectors, the transcriptional co-regulators YAP and TAZ. Pathological YAP and TAZ hyperactivity consequently cause cancer, fibrosis and developmental defects. The Hippo pathway controls an array of fundamental cellular processes, including adhesion, migration, mitosis, polarity and secretion of a range of biologically active components. Recent studies highlight that spatio-temporal regulation of Hippo pathway components are central to precisely controlling its context-dependent dynamic activity. Several levels of feedback are integrated into the Hippo pathway, which is further synergized with interactors outside of the pathway that directly regulate specific Hippo pathway components. Likewise, Hippo core kinases also ‘moonlight’ by phosphorylating multiple substrates beyond the Hippo pathway and thereby integrates further flexibility and robustness in the cellular decision-making process. This topic is still in its infancy but promises to reveal new fundamental insights into the cellular regulation of this therapeutically important pathway. We here highlight recent advances emphasising feedback dynamics and multilevel regulation of the Hippo pathway with a focus on mitosis and cell migration, as well as discuss potential productive future research avenues that might reveal novel insights into the overall dynamics of the pathway.

The HIV co-receptors, CCR5 and CXCR4, are necessary for HIV entry into target cells, interacting with the HIV envelope protein, gp120, to initiate several signaling cascades thought to be important to the entry process. Co-receptor signaling may also promote the development of neuroHIV by contributing to both persistent neuroinflammation and indirect neurotoxicity. But despite the critical importance of CXCR4 and CCR5 signaling to HIV pathogenesis, there is only one therapeutic (the CCR5 inhibitor Maraviroc) that targets these receptors. Moreover, our understanding of co-receptor signaling in the specific context of neuroHIV is relatively poor. Research into co-receptor signaling has largely stalled in the past decade, possibly owing to the complexity of the signaling cascades and functions mediated by these receptors. Examining the many signaling pathways triggered by co-receptor activation has been challenging due to the lack of specific molecular tools targeting many of the proteins involved in these pathways and the wide array of model systems used across these experiments. Studies examining the impact of co-receptor signaling on HIV neuropathogenesis often show activation of multiple overlapping pathways by similar stimuli, leading to contradictory data on the effects of co-receptor activation. To address this, we will broadly review HIV infection and neuropathogenesis, examine different co-receptor mediated signaling pathways and functions, then discuss the HIV mediated signaling and the differences between activation induced by HIV and cognate ligands. We will assess the specific effects of co-receptor activation on neuropathogenesis, focusing on neuroinflammation. We will also explore how the use of substances of abuse, which are highly prevalent in people living with HIV, can exacerbate the neuropathogenic effects of co-receptor signaling. Finally, we will discuss the current state of therapeutics targeting co-receptors, highlighting challenges the field has faced and areas in which research into co-receptor signaling would yield the most therapeutic benefit in the context of HIV infection. This discussion will provide a comprehensive overview of what is known and what remains to be explored in regard to co-receptor signaling and HIV infection, and will emphasize the potential value of HIV co-receptors as a target for future therapeutic development.

For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype. ispartof: JOURNAL OF PROTEOME RESEARCH vol:20 issue:8 pages:3840-3852 ispartof: location:United States status: published

Background Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis. Results We uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited. Conclusions We show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

The chemokine system plays a fundamental role in a diverse range of physiological processes, such as homeostasis and immune responses. Dysregulation in the chemokine system has been linked to inflammatory diseases and cancer, which renders chemokine receptors to be considered as therapeutic targets. In the past two decades, around 45 drugs targeting chemokine receptors have been developed, yet only three are clinically approved. The challenging factors include the limited understanding of aberrant chemokine signalling in malignant diseases, high redundancy of the chemokine system, differences between cell types and non-specific binding of the chemokine receptor antagonists due to the broad ligand-binding pockets. In recent years, emerging studies attempt to characterise the chemokine ligand–receptor interactions and the downstream signalling protein–protein interactions, aiming to fine tuning to the promiscuous interplay of the chemokine system for the development of precision medicine. This review will outline the updates on the mechanistic insights in the chemokine system and propose some potential strategies in the future development of targeted therapy.

Aside from the first week postnatal, murine heart regeneration is restricted and responses to damage follow classic fibrotic remodeling. Recent transcriptomic analyses have suggested that significant cross talk with the sterile immune response could maintain a more embryonic-like signaling network that promotes acute, transient responses. However, with age, this response—likely mediated by neonatal yolk sac macrophages—then transitions to classical macrophage-mediated, cardiac fibroblast (CF)-based remodeling of the extracellular matrix (ECM) after myocardial infarction (MI). The molecular mechanisms that govern the change with age and drive fibrosis via inflammation are poorly understood. Using multiple ribonucleic acid sequencing (RNA-Seq) datasets, we attempt to resolve the relative contributions of CFs and macrophages in the bulk-healing response of regenerative (postnatal day 1) and nonregenerative hearts (postnatal day 8+). We performed an analysis of bulk RNA-Seq datasets from myocardium and cardiac fibroblasts as well as a single-cell RNA-Seq dataset from cardiac macrophages. MI-specific pathway differences revealed that nonregenerative hearts generated more ECM and had larger matricellular responses correlating with inflammation, produced greater chemotactic gradients to recruit macrophages, and expressed receptors for danger-associated molecular patterns at higher levels than neonates. These changes could result in elevated stress-response pathways compared with neonates, converging at NF-κB and activator protein-1 (AP-1) signaling. Profibrotic gene programs, which greatly diverge on day 3 post MI, lay the foundation for chronic fibrosis, and thus postnatal hearts older than 7 days typically exhibit significantly less regeneration. Our analyses suggest that the macrophage ontogenetic shift in the heart postnatally could result in detrimental stress signaling that suppresses regeneration. NEW & NOTEWORTHY Immediately postnatal mammalian hearts are able to regenerate after infarction, but the cells, pathways, and molecules that regulate this behavior are unclear. By comparing RNA-Seq datasets from regenerative mouse hearts and older, nonregenerative hearts, we are able to identify biological processes that are hallmarks of regeneration. We find that sterile inflammatory processes are upregulated in nonregenerative hearts, initiating profibrotic gene programs 3 days after myocardial infarction that can cause myocardial disease. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/macrophage-regulation-of-matrix-remodeling/.

RUNX proteins have been shown to behave as "double-edge sword" in wide variety of cancers. Discovery of non-coding RNAs has played linchpin role in improving our understanding about the post-transcriptional regulation of different cell signaling pathways. Several new mechanistic insights and distinct modes of cross-regulation of RUNX proteins and non-coding RNAs have been highlighted by recent research. In this review we have attempted to provide an intricate interplay between non-coding RNAs and RUNX proteins in different cancers. Better conceptual and mechanistic understanding of layered regulation of RUNX proteins by non-coding RNAs will be helpful in effective translation of the laboratory findings to clinically effective therapeutics.

All physiological events in living organisms originated as specific chemical/biochemical signals on the cell surface and transmitted into the cytoplasm. This signal is translated within milliseconds–hours to a specific and unique order required to maintain optimum performance and homeostasis of living organisms. Examples of daily biological functions include neuronal communication and neurotransmission in the process of learning and memory, secretion (hormones, sweat, and saliva), muscle contraction, cellular growth, differentiation and migration during wound healing, and immunity to fight infections. Among the different transducers for such life-dependent signals is the large family of G protein-coupled receptors (GPCRs). GPCRs constitute roughly 800 genes, corresponding to 2% of the human genome. While GPCRs control a plethora of pathophysiological disorders, only approximately one-third of GPCR families have been deorphanized and characterized. Recent drug data show that around 40% of the recommended drugs available in the market target mainly GPCRs. In this review, we presented how such system signals, either through G protein or via other players, independent of G protein, function within the biological system. We also discussed drugs in the market or clinical trials targeting mainly GPCRs in various diseases, including cancer.

Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through diverse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling.

Cancer is initiated largely by specific cohorts of genetic aberrations, which are generated by mutagens and often mimic active growth factor receptors, or downstream effectors. Once initiated cells outgrow and attract blood vessels, a multi-step process, called metastasis, disseminates cancer cells primarily through vascular routes. The major steps of the metastatic cascade comprise intravasation into blood vessels, circulation as single or collectives of cells, and eventual colonization of distant organs. Herein, we consider metastasis as a multi-step process that seized principles and molecular players employed by physiological processes, such as tissue regeneration and migration of neural crest progenitors. Our discussion contrasts the irreversible nature of mutagenesis, which establishes primary tumors, and the reversible epigenetic processes (e.g. epithelial–mesenchymal transition) underlying the establishment of micro-metastases and secondary tumors. Interestingly, analyses of sequencing data from untreated metastases inferred depletion of putative driver mutations among metastases, in line with the pivotal role played by growth factors and epigenetic processes in metastasis. Conceivably, driver mutations may not confer the same advantage in the microenvironment of the primary tumor and of the colonization site, hence phenotypic plasticity rather than rigid cellular states hardwired by mutations becomes advantageous during metastasis. We review the latest reported examples of growth factors harnessed by the metastatic cascade, with the goal of identifying opportunities for anti-metastasis interventions. In summary, because the overwhelming majority of cancer-associated deaths are caused by metastatic disease, understanding the complexity of metastasis, especially the roles played by growth factors, is vital for preventing, diagnosing and treating metastasis.

Background Multicellular organisms are characterized by a wide diversity of forms and complexity despite a restricted set of key molecules and mechanisms at the base of organismal development. Development combines three basic processes—asymmetric cell division, signaling, and gene regulation—in a multitude of ways to create this overwhelming diversity of multicellular life forms. Here, we use a generative model to test the limits to which such processes can be combined to generate multiple differentiation paths during development, and attempt to chart the diversity of multicellular organisms generated. Results We sample millions of biologically feasible developmental schemes, allowing us to comment on the statistical properties of cell differentiation trajectories they produce. We characterize model-generated “organisms” using the graph topology of their cell type lineage maps. Remarkably, tree-type lineage differentiation maps are the rarest in our data. Additionally, a majority of the “organisms” generated by our model appear to be endowed with the ability to regenerate using pluripotent cells. Conclusions Our results indicate that, in contrast to common views, cell type lineage graphs are unlikely to be tree-like. Instead, they are more likely to be directed acyclic graphs, with multiple lineages converging on the same terminal cell type. Furthermore, the high incidence of pluripotent cells in model-generated organisms stands in line with the long-standing hypothesis that whole body regeneration is an epiphenomenon of development. We discuss experimentally testable predictions of our model and some ways to adapt the generative framework to test additional hypotheses about general features of development.

The salt-inducible kinases, SIK1, SIK2 and SIK3, most closely resemble the AMP-activated protein kinase (AMPK) and other AMPK-related kinases, and like these family members they require phosphorylation by LKB1 to be catalytically active. However, unlike other AMPK-related kinases they are phosphorylated by cyclic AMP-dependent protein kinase (PKA), which promotes their binding to 14-3-3 proteins and inactivation. The most well-established substrates of the SIKs are the CREB-regulated transcriptional co-activators (CRTCs), and the Class 2a histone deacetylases (HDAC4/5/7/9). Phosphorylation by SIKs promotes the translocation of CRTCs and Class 2a HDACs to the cytoplasm and their binding to 14-3-3s, preventing them from regulating their nuclear binding partners, the transcription factors CREB and MEF2. This process is reversed by PKA-dependent inactivation of the SIKs leading to dephosphorylation of CRTCs and Class 2a HDACs and their re-entry into the nucleus. Through the reversible regulation of these substrates and others that have not yet been identified, the SIKs regulate many physiological processes ranging from innate immunity, circadian rhythms and bone formation, to skin pigmentation and metabolism. This review summarises current knowledge of the SIKs and the evidence underpinning these findings, and discusses the therapeutic potential of SIK inhibitors for the treatment of disease.

BackgroundAsexually reproducing populations of single cells evolve through mutation, natural selection, and genetic drift. Environmental conditions in which the evolution takes place define the emergent fitness landscapes. In this work, we used Avida—a digital evolution framework—to uncover a hitherto unexplored interaction between mutation rates, population size, and the relative abundance of metabolizable resources, and its effect on evolutionary outcomes in small populations of digital organisms.ResultsOver each simulation, the population evolved to one of several states, each associated with a single dominant phenotype with its associated fitness and genotype. For a low mutation rate, acquisition of fitness by organisms was accompanied with, and dependent on, an increase in rate of genomic replication. At an increased mutation rate, phenotypes with high fitness values were similarly achieved through enhanced genome replication rates. In addition, we also observed the frequent emergence of suboptimal fitness phenotype, wherein neighboring organisms signaled to each other information relevant to performing metabolic tasks. This metabolic signaling was vital to fitness acquisition and was correlated with greater genotypic and phenotypic heterogeneity in the population. The frequency of appearance of signaling populations increased with population size and with resource abundance.ConclusionsOur results reveal a minimal set of environment–genotype interactions that lead to the emergence of metabolic signaling within evolving populations.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C–X–C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.

Summary Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we investigated the functioning of a cardiomyogenic cell-surface marker, latrophilin-2 (LPHN2), an adhesion G-protein-coupled receptor, in cardiac differentiation. LPHN2 was selectively expressed in cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation; cell sorting with an anti-LPHN2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knockdown or knockout PSCs did not express cardiac genes. We used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways, to assess molecular mechanisms underlying LPHN2-induced cardiac differentiation. LPHN2-dependent phosphorylation was the strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of LPHN2 signaling. These findings provide a valuable strategy for isolating CPCs and CMCs from PSCs and insights into the still-unknown cardiac differentiation mechanisms., Graphical abstract, Highlights • LPHN2 is a cell-surface marker used to separate mouse PSC-derived CPCs and CMCs • CDK5 is a crucial LPHN2 signaling molecule and with Src induces P38MAPK phosphorylation • LPHN2+ cells differentiate to human multipotent CPCs, In this article, Kim and colleagues demonstrate latrophilin-2 (LPHN2) to be a functionally significant marker of mouse and human cardiac differentiation. Further analysis indicates that CDK5 is downstream of LPHN2 and interacts with Src kinase to induce P38MAPK phosphorylation, subsequently activating cardiac-specific gene transcription. LPHN2 can be pivotal to achieving cardiac lineage differentiation, facilitating the clinical application of stem cells in cardiovascular disease.