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HOPS/Tmub1 is a ubiquitously expressed transmembrane ubiquitin-like protein that shuttles between nucleus and cytoplasm during cell cycle progression. HOPS causes cell cycle arrest in G0/G1 phase, an event associated to stabilization of p19Arf, an important tumor suppressor protein. Moreover, HOPS plays an important role in driving centrosomal assembly and maintenance, mitotic spindle proper organization, and ultimately a correct cell division. Recently, HOPS has been described as an important regulator of p53, which acts as modifier, stabilizing p53 half-life and playing a key role in p53 mediating apoptosis after DNA damage. NF-κB is a transcription factor with a central role in many cellular events, including inflammation and apoptosis. Our experiments demonstrate that the transcriptional activity of the p65/RelA NF-κB subunit is regulated by HOPS. Importantly, Hops−/− cells have remarkable alterations of pro-inflammatory responses. Specifically, we found that HOPS enhances NF-κB activation leading to increase transcription of inflammatory mediators, through the reduction of IκBα stability. Notably, this effect is mediated by a direct HOPS binding to the E3 ubiquitin ligase TRAF6, which lessens TRAF6 stability ultimately leading increased IKK complex activation. These findings uncover a previously unidentified function of HOPS/Tmub1 as a novel modulator of TRAF6, regulating inflammatory responses driven by activation of the NF-κB signaling pathway. The comprehension on how HOPS/Tmub1 takes part to the inflammatory processes in vivo and whether this function is important in the control of proliferation and tumorigenesis could establish the basis for the development of novel pharmacological strategies.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice–compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen–matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

The ability to predict invasive fungal infections (IFI) in patients with hematological malignancies is fundamental for successful therapy. Although gut dysbiosis is known to occur in hematological patients, whether airway dysbiosis also contributes to the risk of IFI has not been investigated. Nasal and oropharyngeal swabs were collected for functional microbiota characterization in 173 patients with hematological malignancies recruited in a multicenter, prospective, observational study and stratified according to the risk of developing IFI. A lower microbial richness and evenness were found in the pharyngeal microbiota of high-risk patients that were associated with a distinct taxonomic and metabolic profile. A murine model of IFI provided biologic plausibility for the finding that loss of protective anaerobes, such as Clostridiales and Bacteroidetes, along with an apparent restricted availability of tryptophan, is causally linked to the risk of IFI in hematologic patients and indicates avenues for antimicrobial stewardship and metabolic reequilibrium in IFI. This work was supported by FunMeta Project (ERC-2011-AdG 293714), MicroTher(ERC-2018-PoC-813099), and Gilead (IN-IT-131-4525-518872.9) to L.R. and the GrantAgency of the Czech Republic (GACR No 17-24592Y) and the Czech Ministry ofEducation, Youth and Sports (CETOCOEN PLUS CZ.02.1.01/0.0/0.0/15_003/0000469;LM2015051 and CETOCOEN EXCELLENCE Teaming 2 project CZ.02.1.01/0.0/0.0/18_046/0015975; and Horizon2020 project 857560) to Z.S.

Inflammasomes are powerful cytosolic sensors of environmental stressors and are critical for triggering interleukin-1 (IL-1)-mediated inflammatory responses. However, dysregulation of inflammasome activation may lead to pathological conditions, and the identification of negative regulators for therapeutic purposes is increasingly being recognized. Anakinra, the recombinant form of the IL-1 receptor antagonist, proved effective by preventing the binding of IL-1 to its receptor, IL-1R1, thus restoring autophagy and dampening NLR family pyrin domain containing 3 (NLRP3) activity. As the generation of mitochondrial reactive oxidative species (ROS) is a critical upstream event in the activation of NLRP3, we investigated whether anakinra would regulate mitochondrial ROS production. By profiling the activation of transcription factors induced in murine alveolar macrophages, we found a mitochondrial antioxidative pathway induced by anakinra involving the manganese-dependent superoxide dismutase (MnSOD) or SOD2. Molecularly, anakinra promotes the binding of SOD2 with the deubiquitinase Ubiquitin Specific Peptidase 36 (USP36) and Constitutive photomorphogenesis 9 (COP9) signalosome, thus increasing SOD2 protein longevity. Functionally, anakinra and SOD2 protects mice from pulmonary oxidative inflammation and infection. On a preclinical level, anakinra upregulates SOD2 in murine models of chronic granulomatous disease (CGD) and cystic fibrosis (CF). These data suggest that protection from mitochondrial oxidative stress may represent an additional mechanism underlying the clinical benefit of anakinra and identifies SOD2 as a potential therapeutic target.

Inflammation plays a major role in the pathophysiology of cystic fibrosis (CF), a multisystem disease. Anti-inflammatory therapies are, therefore, of interest in CF, provided that the inhibition of inflammation does not compromise the ability to fight pathogens. Here, we assess whether indole-3-aldehyde (3-IAld), a ligand of the aryl hydrocarbon receptor (AhR), may encompass such an activity. We resorted to biopharmaceutical technologies in order to deliver 3-IAld directly into the lung, via dry powder inhalation, or into the gut, via enteric microparticles, in murine models of CF infection and inflammation. We found the site-specific delivery of 3-IAld to be an efficient strategy to restore immune and microbial homeostasis in CF organs, and mitigate lung and gut inflammatory pathology in response to fungal infections, in the relative absence of local and systemic inflammatory toxicity. Thus, enhanced delivery to target organs of AhR agonists, such as 3-IAld, may pave the way for the development of safe and effective anti-inflammatory agents in CF.

The circadian clock driven by the daily light–dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other’s expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding for the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Long considered a lung disease for the devastating impact on the respiratory function, the recent diagnostic and therapeutic advances have shed the light on the extra-pulmonary manifestations of CF, including gastrointestinal, hepatobiliary and pancreatic symptoms. We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease.

The organisms of most domains of life have adapted to circadian changes of the environment and regulate their behavior and physiology accordingly. A particular case of such paradigm is represented by some types of host-pathogen interaction during infection. Indeed, not only some hosts and pathogens are each endowed with their own circadian clock, but they are also influenced by the circadian changes of the other with profound consequences on the outcome of the infection. It comes that daily fluctuations in the availability of resources and the nature of the immune response, coupled with circadian changes of the pathogen, may influence microbial virulence, level of colonization and damage to the host, and alter the equilibrium between commensal and invading microorganisms. In the present review, we discuss the potential relevance of circadian rhythms in human bacterial and fungal pathogens, and the consequences of circadian changes of the host immune system and microbiome on the onset and development of infection. By looking from the perspective of the interplay between host and microbes circadian rhythms, these concepts are expected to change the way we approach human infections, not only by predicting the outcome of the host-pathogen interaction, but also by indicating the best time for intervention to potentiate the anti-microbial activities of the immune system and to weaken the pathogen when its susceptibility is higher.

Cystic fibrosis (CF) is a rare genetic disorder caused by a defect in the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR), resulting in ionic imbalance of surface fluid. Although affecting multiple organs, the progressive deterioration of respiratory function by recurrent infections and chronic inflammation represents the main cause of morbidity and mortality in CF patients. The development of modulators targeting the basic defect of CFTR has represented a major breakthrough in CF therapy, but the impact on inflammation has remained enigmatic. The emerging scenario taking hold in the field points to inflammation as a major, somehow missed, therapeutic target for prevention of lung decline. Not surprisingly, the development of anti-inflammatory drugs is taking its share in the drug development pipeline. But the path is not straightforward and targeting inflammation should be balanced with the increased risk of infection. The strategy to restore the homeostatic regulation of inflammation to efficiently respond to infection while preventing lung damage needs to be based on identifying and targeting endogenous immunoregulatory pathways that are defective in CF. We herein provide an overview of anti-inflammatory drugs currently approved or under investigation in CF patients, and present our recent studies on how the knowledge on defective immune pathways in CF may translate into innovative and selective anti-inflammatory therapeutics. Through the discovery of naturally occurring molecules or their synthetic mimics, this review emphasizes the critical importance of selectively targeting key inflammatory pathways to preserve immunocompetence in CF patients.

The innate immune system represents the host’s first-line defense against pathogens, dead cells or environmental factors. One of the most important inflammatory pathways is represented by the activation of the NOD-like receptor (NLR) protein family. Some NLRs induce the assembly of large caspase-1-activating complexes called inflammasomes. Different types of inflammasomes have been identified that can respond to distinct bacterial, viral or fungal infections; sterile cell damage or other stressors, such as metabolic imbalances. Epigenetic regulation has been recently suggested to provide a complementary mechanism to control inflammasome activity. This regulation can be exerted through at least three main mechanisms, including CpG DNA methylation, histones post-translational modifications and noncoding RNA expression. The repression or promotion of expression of different inflammasomes (NLRP1, NLRP2, NLRP3, NLRP4, NLRP6, NLRP7, NLRP12 and AIM2) through epigenetic mechanisms determines the development of pathologies with variable severity. For example, our team recently explored the role of microRNAs (miRNAs) targeting and modulating the components of the inflammasome as potential biomarkers in bladder cancer and during therapy. This suggests that the epigenetic control of inflammasome-related genes could represent a potential target for further investigations of molecular mechanisms regulating inflammatory pathways.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1–/– mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride-channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF —which include impaired chloride permeability and persistent lung inflammation—a multidrug approach is required for efficacious CF therapy. To date, no individual, drug with pleiotropic beneficial effects for CF is available. Here we report on the ability of thymosin alpha 1 (Tα1)—a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent—to rectify the multiple tissue defects in CF mice as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology; namely, it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 offers a strong potential to be an efficacious single molecule-based therapeutic agent in CF.

This study demonstrates that thymosin β4 stabilizes HIF-1a to promote autophagy and up-regulate genes involved in tissue and mucosal barrier protection in chronic granulomatous disease., Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin β4 (Tβ4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tβ4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tβ4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tβ4.

Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida spp., most frequently by Candida albicans, which may become recurrent and severely impacting the quality of life of susceptible women. Although it is increasingly being recognized that mucosal damage is mediated by an exaggerated inflammatory response, current therapeutic approaches are only based on antifungals that may relieve the symptomatology, but fail to definitely prevent recurrences. The unrestrained activation of the NLRP3 inflammasome with continuous production of IL-1β and recruitment of neutrophils is recognized as a pathogenic factor in VVC. We have previously shown that IL-22 is required to dampen pathogenic inflammasome activation in VVC via the NLRC4/IL-1Ra axis. However, IL-22 also regulates IL-18, a product of the inflammasome activity that regulates IL-22 expression. Here we describe a cross-regulatory circuit between IL-18 and IL-22 in murine VVC that is therapeutically druggable. We found that IL-18 production was dependent on IL-22 and NLRC4, and that IL-18, in turn, contributes to IL-22 activity. Like in IL-22 deficiency, IL-18 deficiency was associated with an increased susceptibility to VVC and unbalanced Th17/Treg response, suggesting that IL-18 can regulate both the innate and the adaptive responses to the fungus. Administration of the microbial metabolite indole-3-aldehyde, known to stimulate the production of IL-22 via the aryl hydrocarbon receptor (AhR), promoted IL-18 expression and protection against Candida infection. Should low levels of IL-18 be demonstrated in the vaginal fluids of women with recurrent VVC, targeting the AhR/IL-22/IL-18 pathway could be exploited for future therapeutic approaches in VVC. This study suggests that a deeper understanding of the mechanisms regulating inflammasome activity may lead to the identification of novel targets for intervention in VVC.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of gluten and characterized by reversible small-bowel mucosal atrophy in genetically predisposed subjects. Although the prevalence of CD has increased, many aspects of this pathology are still unrecognized. Candida albicans, a commensal of the human gastrointestinal tract, has been linked to CD for a long time based, among others, upon the observation of similarity between the fungal wall component, hyphal wall protein 1, and CD-related gliadin T-cell epitopes. We have recently demonstrated that Candida may switch from commensal to pathogen contingent upon several players, including mast cells, key sentinels of the immune system at the interface between the environment and the host, and the pleiotropic cytokine IL-9. However, other factors are likely to play a role by altering the balance between inflammation and tolerance. In this regard, tryptophan and its metabolites are increasingly being recognized in promoting mucosal homeostasis by balancing the immune response to external cues. Based on these premises, we will discuss how the output of Candida colonization in the gut is highly contextual, being determined at the intersection of many immunological (IL-9/mast cells) and metabolic (tryptophan) pathways that ultimately dictate the Candida commensalism vs. pathogenicity in CD, thus paving the way for novel therapeutic opportunities in CD.

Liver regeneration offers a distinctive opportunity to study cell proliferation in vivo. Mammalian silent information regulator 1 (SIRT1), a NAD+-dependent histone deacetylase, is an important regulator of various cellular processes, including proliferation, metabolism, and circadian rhythms. In the liver, SIRT1 coordinates the circadian oscillation of clock-controlled genes, including genes that encode enzymes involved in metabolic pathways. We performed partial hepatectomy in WT and liver-specific Sirt1-deficient mice and analyzed the expression of cell cycle regulators in liver samples taken at different times during the regenerative process, by real time PCR, Western blotting analysis, and immunohistochemistry. Lipidomic analysis was performed in the same samples by MS/HPLC. We showed that G1/S progression was significantly affected by absence of SIRT1 in the liver, as well as circadian gene expression. This was associated to lipid accumulation due to defective fatty acid beta-oxidation. Our study revealed for the first time the importance of SIRT1 in the regulation of hepatocellular proliferation, circadian rhythms, and lipid metabolism during liver regeneration in mice. These results represent an additional step toward the characterization of SIRT1 function in the liver.

This study demonstrates that Tα1 protects mice from anti–CTLA-4–induced colitis and sustains its antitumor activity, thus suggesting that Tα1 may be used in combination protocols., The advent of immune checkpoint inhibitors has represented a major boost in cancer therapy, but safety concerns are increasingly being recognized. Indeed, although beneficial at the tumor site, unlocking a safeguard mechanism of the immune response may trigger autoimmune-like effects at the periphery, thus making the safety of immune checkpoint inhibitors a research priority. Herein, we demonstrate that thymosin α1 (Tα1), an endogenous peptide with immunomodulatory activities, can protect mice from intestinal toxicity in a murine model of immune checkpoint inhibitor–induced colitis. Specifically, Tα1 efficiently prevented immune adverse pathology in the gut by promoting the indoleamine 2,3-dioxygenase (IDO) 1–dependent tolerogenic immune pathway. Notably, Tα1 did not induce IDO1 in the tumor microenvironment, but rather modulated the infiltration of T-cell subsets by inverting the ratio between CD8+ and Treg cells, an effect that may depend on Tα1 ability to regulate the differentiation and chemokine expression profile of DCs. Thus, through distinct mechanisms that are contingent upon the context, Tα1 represents a plausible candidate to improve the safety/efficacy profile of immune checkpoint inhibitors.

Organisms adapt to day–night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium ( S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. We previously reported that silent information regulator 1 (SIRT1), a NAD + -dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD + . Here we used specific SIRT1 activator compounds both in vitro and in vivo. We tested a variety of compounds to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. Our findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications.

The liver is the most important organ in cholesterol metabolism, which is instrumental in regulating cell proliferation and differentiation. The gene Tm7sf2 codifies for 3 β-hydroxysterol-Δ14-reductase (C14-SR), an endoplasmic reticulum resident protein catalyzing the reduction of C14-unsaturated sterols during cholesterol biosynthesis from lanosterol. In this study we analyzed the role of C14-SR in vivo during cell proliferation by evaluating liver regeneration in Tm7sf2 knockout (KO) and wild-type (WT) mice. Tm7sf2 KO mice showed no alteration in cholesterol content. However, accumulation and delayed catabolism of hepatic triglycerides was observed, resulting in persistent steatosis at all times post hepatectomy. Moreover, delayed cell cycle progression to the G1/S phase was observed in Tm7sf2 KO mice, resulting in reduced cell division at the time points examined. This was associated to abnormal ER stress response, leading to alteration in p53 content and, consequently, induction of p21 expression in Tm7sf2 KO mice. In conclusion, our results indicate that Tm7sf2 deficiency during liver regeneration alters lipid metabolism and generates a stress condition, which, in turn, transiently unbalances hepatocytes cell cycle progression.

Mammalian sirtuins and the circadian clock appear to be intimately linked. The circadian clock is able to modulate the activity of SIRT1 while SIRT1 also deacetylates specific elements of the clock machinery and modifies histones to regulate gene expression. In this chapter, we review the current understanding of the field on the interrelationship between SIRT1 and the circadian clock machinery, how this regulation occurs, and what other possible functions the sirtuins could have in circadian biology. This chapter also discusses the critical metabolic cues that are in place to regulate enzymes involved in histone modifications and subsequent regulation of gene expression. The role of the clock in regulating metabolic state of the cell is also a concept that will be explored, as the clock-dependent control over metabolism is far more extensive than originally thought. The concept that metabolism and epigenetics are closely linked opens new directions that need to be explored in terms of circadian metabolic regulation and how this modulates a host of epigenetic modifying enzymes and chromatin remodelers.

Circadian rhythms regulate a wide variety of physiological and metabolic processes. The clock machinery comprises complex transcriptional–translational feedback loops that, through the action of specific transcription factors, modulate the expression of as many as 10% of cellular transcripts. This marked change in gene expression necessarily implicates a global regulation of chromatin remodeling. Indeed, various descriptive studies have indicated that histone modifications occur at promoters of clock-controlled genes (CCGs) in a circadian manner. The finding that CLOCK, a transcription factor crucial for circadian function, has intrinsic histone acetyl transferase (HAT) activity has paved the way to unraveling the molecular mechanisms that govern circadian chromatin remodeling. A search for the histone deacetylase (HDAC) that counterbalances CLOCK activity revealed that SIRT1, a nicotinamide adenin dinucleotide (NAD+)-dependent HDAC, functions in a circadian manner. Importantly, SIRT1 is a regulator of aging, inflammation and metabolism. As many transcripts that oscillate in mammalian peripheral tissues encode proteins that have central roles in metabolic processes, these findings establish a functional and molecular link between energy balance, chromatin remodeling and circadian physiology. Here we review recent studies that support the existence of this link and discuss their implications for understanding mammalian physiology and pathology.

Background/Aims: Transcription factors coupled to cyclic adenosine mono phosphate (cAMP) signalling in the cAMP-responsive elements binding (CREB)/ATF family constitute a family of activators or repressors that bind to cAMP-responsive promoter elements (CREs) in the regulatory regions of cAMP-inducible genes. A role for CREB/ATF family has been advocated in the control of hepatocellular carcinoma progression. CREB appears to be activated by the X protein of hepatitis B virus, which links to the unphosphorylated form of CREB and activates transcription, thus obviating an otherwise indispensable Ser-133 phosphorylation. Identification of factors capable of triggering transcription via cAMP-responsive elements modulator (CREM)/CREB signalling in the absence of Ser phosphorylation will improve our knowledge of the molecular mechanism of liver cell proliferation. Methods: To isolate and study proteins binding and activating CREB and/or CREM in the liver, we performed the screening of a mouse liver cDNA library using the Two-Hybrid System. Results: We report the identification and characterization of a novel peptide, VTIP-peptide (VTIP-P), which binds and enhances the activation of CREM/CREB, obviating the need for transcription factor phosphorylation. We demonstrated that VTIP-P physically interacts with the activation domain (AD) of the transcription factors CREB/CREM and activates transcription by modifying their phosphorylation pattern in hepatoma cells. The data allowed the conclusion that VTIP-P binds the AD of CREB and CREM by stabilizing their phosphorylation. Conclusion: The characterization of molecules capable of interfering in the liver with an important pathway such as CREB could be significant in designing and/or developing new therapeutic approaches to the control of liver cell proliferation.

Liver regeneration is a unique means of studying cell proliferation in vivo. Screening of a large cDNA library from regenerating liver has previously allowed us to identify and characterize a cluster of genes encoding proteins with important roles in proliferative processes. Here, by examining different rat and human tissues as well as cell lines, we characterized a highly conserved gene, guanylyl cyclase domain containing 1 (GUCD1), whose modulation occurs in liver regeneration and cell cycle progression in vitro. High-level expression of GUCD1 transcripts was observed in livers from patients with hepatocellular carcinoma. A yeast two-hybrid interaction assay, aimed at identifying any relevant interaction partners of GUCD1, revealed direct interactions with NEDD4-1 (E3 ubiquitin protein ligase neural precursor cell expressed, developmentally downregulated gene 4), resulting in control of GUCD1 stability. Thus, we have characterized expression and function of a ubiquitous protein, GUCD1, which might have a role in regulating normal and abnormal cell growth in the liver.

Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases.

Hepatocyte odd protein shuttling (HOPS) moves between nucleus and cytoplasm. HOPS overexpression leads to cell cycle arrest in G 0/G 1, and HOPS knockdown causes centrosome alterations, with subsequent abnormal cell division. Recently, we demonstrated that HOPS acts as a functional bridge in NPM-p19(Arf) interactions. Here we show that HOPS is present in 3 different isoforms that play distinct intracellular functions. Although HOPS is a transmembrane ubiquitin, an isoform with intermediate molecular weight is cleaved from the membrane and released into the cytosol, to act as the shuttling protein. We identified a signal peptide peptidase structure in N-terminal membrane-bound HOPS that allows the regulated intramembrane proteolysis (RIP) system to control the relative amounts of the released, shuttling isoform capable of binding NPM. These results argue for distinct, isoform-specific functions of HOPS in the nucleolus, nucleus, and cytoplasm and provide insight into the dynamics of HOPS association with NPM, whose mutation and subsequent delocalization is found in 30% of acute myeloid leukemia patients.

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

The circadian system controls a large array of physiological and metabolic functions. The molecular organization of the circadian clock is complex, involving various elements organized in feedback regulatory loops. Here we demonstrate that the RelB subunit of NFκB acts as a repressor of circadian transcription. RelB physically interacts with the circadian activator BMAL1 in the presence of CLOCK to repress circadian gene expression at the promoter of the clock-controlled gene Dbp. The repression is independent of the circadian negative regulator CRY. Notably, RelB-/-fibroblasts have profound alterations of circadian genes expression. These findings reveal a previously unforeseen function for RelB as an important regulator of the mammalian circadian system in fibroblasts. © 2012 Landes Bioscience.

The mammalian cell contains a molecular clock that contributes, within each organism, to circadian rhythms and variety of physiological and metabolic processes. The clock machinery is constituted by interwined transcriptional-translational feedback loops that, through the action of specific transcription factors, modulate the expression of clock-controlled genes. These oscillations in gene expression necessarily implicate events of chromatin remodeling on a relatively large, global scale, considering that as many 10% of cellular transcripts oscillate in a circadian manner. CLOCK, a transcription factor crucial for circadian function, has intrinsic histone acetyltransferase activity and operates within a large nuclear complex with other chromatin remodelers. CLOCK directs the cyclic acetylation of the histone H3 and of its own partner BMAL1. A search for the histone deacetylase (HDAC) that counterbalanced CLOCK activity revealed that SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent HDAC, functions in a circadian manner. Importantly, SIRT1 is a regulator of several metabolic processes and was found to interact with CLOCK and to be recruited to circadian promoters in a cyclic manner. As many transcripts that oscillate in mammalian peripheral tissues encode proteins that have central roles in metabolic processes, these findings establish a functional and molecular link among energy balance, chromatin remodeling, and circadian physiology.

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention. © 2010 Elsevier Inc.

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, gamma-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.

Major mood disorders have been linked to abnormalities in circadian rhythms, leading to disturbances in sleep, mood, temperature, and hormonal levels. We provide evidence that ketamine, a drug with rapid antidepressant effects, influences the function of the circadian molecular machinery. Ketamine modulates CLOCK:BMAL1-mediated transcriptional activation when these regulators are ectopically expressed in NG108-15 neuronal cells. Inhibition occurs in a dose-dependent manner and is attenuated after treatment with the GSK3β antagonist SB21673. We analyzed the effect of ketamine on circadian gene expression and observed a dose-dependent reduction in the amplitude of circadian transcription of the Bmal1, Per2, and Cry1 genes. Finally, chromatin-immunoprecipitation analyses revealed that ketamine altered the recruitment of the CLOCK:BMAL1 complex on circadian promoters in a time-dependent manner. Our results reveal a yet unsuspected molecular mode of action of ketamine and thereby may suggest possible pharmacological antidepressant strategies.