AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance.

AMPK is a highly conserved master regulator of metabolism, which restores energy balance during metabolic stress both at the cellular and physiological levels. The identification of numerous AMPK targets has helped explain how AMPK restores energy homeostasis. Recent advancements illustrate novel mechanisms of AMPK regulation, including changes in subcellular localization and phosphorylation by non-canonical upstream kinases. Notably, the therapeutic potential of AMPK is widely recognized and heavily pursued for treatment of metabolic diseases such as diabetes, but also obesity, inflammation, and cancer. Moreover, the recently solved crystal structure of AMPK has shed light both into how nucleotides activate AMPK and, importantly, also into the sites bound by small molecule activators, thus providing a path for improved drugs.

Insulin-degrading enzyme: an ally against metabolic and neurodegenerative diseases.

Insulin-degrading enzyme (IDE) function goes far beyond its known proteolytic role as a regulator of insulin levels. IDE has a wide substrate promiscuity, degrading several proteins such as amyloid-ß peptide, glucagon, islet amyloid polypeptide (IAPP), and insulin-like growth factors, which have diverse physiological and pathophysiological functions. Importantly, IDE plays other non-proteolytic functions such as: a chaperone/dead-end chaperone, an E1-ubiquitin activating enzyme, and a proteasome modulator. It also responds as a heat shock protein, regulating cellular proteostasis. Notably, amyloidogenic proteins such as IAPP, amyloid-ß, and α-synuclein have been reported as substrates for IDE chaperone activity. This is of utmost importance as failure of IDE may result in increased protein aggregation, a key hallmark in the pathogenesis of beta cells in type 2 diabetes mellitus and of neurons in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this review, we focus on the biochemical and biophysical properties of IDE and the regulation of its physiological functions. We further raise the hypothesis that IDE plays a central role in the pathological context of dysmetabolic and neurodegenerative diseases and discuss its potential as a therapeutic target. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical Presentation, Genetic Etiology, and Coenzyme Q10 Levels in 55 Children with Combined Enzyme Deficiencies of the Mitochondrial Respiratory Chain.

OBJECTIVES: To evaluate the clinical symptoms and biochemical findings and establish the genetic etiology in a cohort of pediatric patients with combined deficiencies of the mitochondrial respiratory chain complexes. STUDY DESIGN: Clinical and biochemical data were collected from 55 children. All patients were subjected to sequence analysis of the entire mitochondrial genome, except when the causative mutations had been identified based on the clinical picture. Whole exome sequencing/whole genome sequencing (WES/WGS) was performed in 32 patients. RESULTS: Onset of disease was generally early in life (median age, 6 weeks). The most common symptoms were muscle weakness, hypotonia, and developmental delay/intellectual disability. Nonneurologic symptoms were frequent. Disease causing mutations were found in 20 different nuclear genes, and 7 patients had mutations in mitochondrial DNA. Causative variants were found in 18 of the 32 patients subjected to WES/WGS. Interestingly, many patients had low levels of coenzyme Q10 in muscle, irrespective of genetic cause. CONCLUSIONS: Children with combined enzyme defects display a diversity of clinical symptoms with varying age of presentation. We established the genetic diagnosis in 35 of the 55 patients (64%). The high diagnostic yield was achieved by the introduction of massive parallel sequencing, which also revealed novel genes and enabled elucidation of new disease mechanisms.

ACE2 and energy metabolism: the connection between COVID-19 and chronic metabolic disorders.

The renin-angiotensin system (RAS) has currently attracted increasing attention due to its potential function in regulating energy homeostasis, other than the actions on cellular growth, blood pressure, fluid, and electrolyte balance. The existence of RAS is well established in metabolic organs, including pancreas, liver, skeletal muscle, and adipose tissue, where activation of angiotensin-converting enzyme (ACE) - angiotensin II pathway contributes to the impairment of insulin secretion, glucose transport, fat distribution, and adipokines production. However, the activation of angiotensin-converting enzyme 2 (ACE2) - angiotensin (1-7) pathway, a novel branch of the RAS, plays an opposite role in the ACE pathway, which could reverse these consequences by improving local microcirculation, inflammation, stress state, structure remolding, and insulin signaling pathway. In addition, new studies indicate the protective RAS arm possesses extraordinary ability to enhance brown adipose tissue (BAT) activity and induces browning of white adipose tissue, and consequently, it leads to increased energy expenditure in the form of heat instead of ATP synthesis. Interestingly, ACE2 is the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is threating public health worldwide. The main complications of SARS-CoV-2 infected death patients include many energy metabolism-related chronic diseases, such as diabetes. The specific mechanism leading to this phenomenon is largely unknown. Here, we summarize the latest pharmacological and genetic tools on regulating ACE/ACE2 balance and highlight the beneficial effects of the ACE2 pathway axis hyperactivity on glycolipid metabolism, as well as the thermogenic modulation.

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

DUSP9, a Dual-Specificity Phosphatase with a Key Role in Cell Biology and Human Diseases.

Mitogen-activated protein kinases (MAPKs) are essential for proper cell functioning as they regulate many molecular effectors. Careful regulation of MAPKs is therefore required to avoid MAPK pathway dysfunctions and pathologies. The mammalian genome encodes about 200 phosphatases, many of which dephosphorylate the MAPKs and bring them back to an inactive state. In this review, we focus on the normal and pathological functions of dual-specificity phosphatase 9 (DUSP9)/MAP kinase phosphatases-4 (MKP-4). This cytoplasmic phosphatase, which belongs to the threonine/tyrosine dual-specific phosphatase family and was first described in 1997, is known to dephosphorylate ERK1/2, p38, JNK and ASK1, and thereby to control various MAPK pathway cascades. As a consequence, DUSP9 plays a major role in human pathologies and more specifically in cardiac dysfunction, liver metabolic syndromes, diabetes, obesity and cancer including drug response and cell stemness. Here, we recapitulate the mechanism of action of DUSP9 in the cell, its levels of regulation and its roles in the most frequent human diseases, and discuss its potential as a therapeutic target.

Novel Inhibitors of Nicotinamide-N-Methyltransferase for the Treatment of Metabolic Disorders.

Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.

Inhibition of ROCK2 alleviates renal fibrosis and the metabolic disorders in the proximal tubular epithelial cells.

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-ß1 (TGF-ß1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-ß1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

Sirtuins and NAD+ in the Development and Treatment of Metabolic and Cardiovascular Diseases.

The sirtuin family of nicotinamide adenine dinucleotide-dependent deacylases (SIRT1-7) are thought to be responsible, in large part, for the cardiometabolic benefits of lean diets and exercise and when upregulated can delay key aspects of aging. SIRT1, for example, protects against a decline in vascular endothelial function, metabolic syndrome, ischemia-reperfusion injury, obesity, and cardiomyopathy, and SIRT3 is protective against dyslipidemia and ischemia-reperfusion injury. With increasing age, however, nicotinamide adenine dinucleotide levels and sirtuin activity steadily decrease, and the decline is further exacerbated by obesity and sedentary lifestyles. Activation of sirtuins or nicotinamide adenine dinucleotide repletion induces angiogenesis, insulin sensitivity, and other health benefits in a wide range of age-related cardiovascular and metabolic disease models. Human clinical trials testing agents that activate SIRT1 or boost nicotinamide adenine dinucleotide levels are in progress and show promise in their ability to improve the health of cardiovascular and metabolic disease patients.

Carnosic acid prevented olanzapine-induced metabolic disorders through AMPK activation.

Olanzapine, an atypical antipsychotic medication, has been associated with weight gain and metabolic toxicity, especially in long term usage. Carnosic acid (CA), a major constituent of rosemary extract, has been shown to improve metabolic abnormalities. In this experiment, the effect of CA on olanzapine-induced obesity and metabolic toxicity has been evaluated. Female Wistar rats were divided into six groups. (1) control; (2) olanzapine (5 mg/kg/day, IP); (3, 4 and 5) olanzapine (5 mg/kg/day, IP) plus CA (5, 10 and 20 mg/kg/day, gavage) and (6) CA (20 mg/kg/day, gavage). Bodyweight and food intake were measured during the study. After 14 days, mean systolic blood pressure (MSBP), glycemia, serum lipid profile, the serum concentration of leptin, insulin, AMPK, P-AMPK, and P-ACC liver protein levels were evaluated. The mean weight in the group received olanzapine increased by 4.8 g at the end of the study. The average food intake was increased by olanzapine. Olanzapine increased triglyceride, fasting blood glucose (FBG), and leptin levels. It increased MSBP and down-regulated P-AMPK/AMPK ratio and P-ACC protein levels. CA (three doses) decreased body weight gain and reduced average food intake at 10 and 20 mg/kg. CA especially at the highest dose decreased the changes in lipid profile, FBG, leptin level, and MSBP. P-AMPK/AMPK and P-ACC protein levels were increased by carnosic acid. In conclusion, the activation of AMPK by CA can be proposed as a key mechanism against olanzapine-induced metabolic toxicity where the activation of AMPK increases fat consumption and regulates glucose hemostasis in the liver.

A Molecular Perspective on Sirtuin Activity.

The protein acetylation of either the α-amino groups of amino-terminal residues or of internal lysine or cysteine residues is one of the major posttranslational protein modifications that occur in the cell with repercussions at the protein as well as at the metabolome level. The lysine acetylation status is determined by the opposing activities of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), which add and remove acetyl groups from proteins, respectively. A special group of KDACs, named sirtuins, that require NAD+ as a substrate have received particular attention in recent years. They play critical roles in metabolism, and their abnormal activity has been implicated in several diseases. Conversely, the modulation of their activity has been associated with protection from age-related cardiovascular and metabolic diseases and with increased longevity. The benefits of either activating or inhibiting these enzymes have turned sirtuins into attractive therapeutic targets, and considerable effort has been directed toward developing specific sirtuin modulators. This review summarizes the protein acylation/deacylation processes with a special focus on the current developments in the sirtuin research field.

Metabolic roles of poly(ADP-ribose) polymerases.

Poly(ADP-ribosyl)ation (PARylation) is an evolutionarily conserved reaction that had been associated with numerous cellular processes such as DNA repair, protein turnover, inflammatory regulation, aging or metabolic regulation. The metabolic regulatory tasks of poly(ADP-ribose) polymerases (PARPs) are complex, it is based on the regulation of metabolic transcription factors (e.g. SIRT1, nuclear receptors, SREBPs) and certain cellular energy sensors. PARP over-activation can cause damage to mitochondrial terminal oxidation, while the inhibition of PARP-1 or PARP-2 can induce mitochondrial oxidation by enhancing the mitotropic tone of gene transcription and signal transduction. These PARP-mediated processes impact on higher order metabolic regulation that modulates lipid metabolism, circadian oscillations and insulin secretion and signaling. PARP-1, PARP-2 and PARP-7 are related to metabolic diseases such as diabetes, alcoholic and non-alcoholic fatty liver disease (AFLD, NAFLD), or on a broader perspective to Warburg metabolism in cancer or the metabolic diseases accompanying aging.

Localization of Human Glutamate Dehydrogenases Provides Insights into Their Metabolic Role and Their Involvement in Disease Processes.

Glutamate dehydrogenase (GDH) catalyzes the reversible deamination of L-glutamate to α-ketoglutarate and ammonia. In mammals, GDH contributes to important processes such as amino acid and carbohydrate metabolism, energy production, ammonia management, neurotransmitter recycling and insulin secretion. In humans, two isoforms of GDH are found, namely hGDH1 and hGDH2, with the former being ubiquitously expressed and the latter found mainly in brain, testis and kidney. These two iso-enzymes display highly divergent allosteric properties, especially concerning their basal activity, ADP activation and GTP inhibition. On the other hand, both enzymes are thought to predominantly localize in the mitochondrial matrix, even though alternative localizations have been proposed. To further study the subcellular localization of the two human iso-enzymes, we created HEK293 cell lines stably over-expressing hGDH1 and hGDH2. In these cell lines, immunofluorescence and enzymatic analyses verified the overexpression of both hGDH1 and hGDH2 iso-enzymes, whereas subcellular fractionation followed by immunoblotting showed their predominantly mitochondrial localization. Given that previous studies have only indirectly compared the subcellular localization of the two iso-enzymes, we co-expressed them tagged with different fluorescent dyes (green and red fluorescent protein for hGDH1 and hGDH2, respectively) and found them to co-localize. Despite the wealth of information related to the functional properties of hGDH1 and hGDH2 and the availability of the hGDH1 structure, there is still an ongoing debate concerning their metabolic role and their involvement in disease processes. Data on the localization of hGDHs, as the ones presented here, could contribute to better understanding of the function of these important human enzymes.

Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic Disease.

RATIONALE: Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. OBJECTIVE: Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. METHODS AND RESULTS: Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (HdbWnox1), lean Nox1 knockout (HdbKnox1), obese (KdbWnox1), and obese KK (KdbKnox1). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW (P<0.01), whereas deletion of Nox1 in KW mice normalized dilation. Vasodilator responses after inhibition of NO synthase blunted acetylcholine responses in KK and lean controls, but had no impact in KW, attributing recovered dilatory capacity in KK to normalization of NO. Acetylcholine responses were improved (P<0.05) with Tempol, and histochemistry revealed oxidative stress in KW animals, whereas Tempol had no impact and reactive oxygen species staining was negligible in KK. Blunted dilatory responses to an NO donor and loss of myogenic tone in KW animals were also rescued with Nox1 deletion. CONCLUSIONS: Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.

An Updated Review of Lysophosphatidylcholine Metabolism in Human Diseases.

Lysophosphatidylcholine (LPC) is increasingly recognized as a key marker/factor positively associated with cardiovascular and neurodegenerative diseases. However, findings from recent clinical lipidomic studies of LPC have been controversial. A key issue is the complexity of the enzymatic cascade involved in LPC metabolism. Here, we address the coordination of these enzymes and the derangement that may disrupt LPC homeostasis, leading to metabolic disorders. LPC is mainly derived from the turnover of phosphatidylcholine (PC) in the circulation by phospholipase A2 (PLA2). In the presence of Acyl-CoA, lysophosphatidylcholine acyltransferase (LPCAT) converts LPC to PC, which rapidly gets recycled by the Lands cycle. However, overexpression or enhanced activity of PLA2 increases the LPC content in modified low-density lipoprotein (LDL) and oxidized LDL, which play significant roles in the development of atherosclerotic plaques and endothelial dysfunction. The intracellular enzyme LPCAT cannot directly remove LPC from circulation. Hydrolysis of LPC by autotaxin, an enzyme with lysophospholipase D activity, generates lysophosphatidic acid, which is highly associated with cancers. Although enzymes with lysophospholipase A1 activity could theoretically degrade LPC into harmless metabolites, they have not been found in the circulation. In conclusion, understanding enzyme kinetics and LPC metabolism may help identify novel therapeutic targets in LPC-associated diseases.

Human hepatocyte transplantation corrects the inherited metabolic liver disorder arginase deficiency in mice.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.

Plasma Xanthine Oxidoreductase Activity as a Novel Biomarker of Metabolic Disorders in a General Population.

BACKGROUND: Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the formation of uric acid from hypoxanthine and xanthine, leading to an increase in superoxide and reactive oxygen species. Activation of XOR promotes oxidative stress-related tissue injury. We investigated the associations between metabolic parameters and plasma XOR activity measured by a sensitive and accurate assay using a combination of liquid chromatography and triple quadrupole mass spectrometry to detect [13C2,15N2]-uric acid using [13C2,15N2]-xanthine as a substrate.Methods and Results:A total of 627 Japanese subjects (M/F, 292/335) from the Tanno-Sobetsu Study, a population-based cohort, were recruited. Plasma XOR activity was significantly higher in males than in females, and habitual smoking was associated with elevation of activity. Plasma XOR activity was positively correlated with body mass index (BMI; r=0.323, P<0.001), waist circumference, blood pressure, and levels of liver enzymes including alanine transaminase (r=0.694, P<0.001), uric acid (r=0.249, P<0.001), triglycerides (r=0.312, P<0.001), hemoglobin A1c, fasting glucose, insulin and HOMA-R (r=0.238, P<0.001) as a marker of insulin resistance and was negatively correlated with high-density lipoprotein cholesterol level. On stepwise and multivariate regression analyses, BMI, smoking and levels of alanine transaminase, uric acid, triglycerides and HOMA-R were independent predictors of plasma XOR activity after adjustment for age and gender. CONCLUSIONS: Plasma XOR activity is a novel biomarker of metabolic disorders in a general population.

Inhibition of HIF-prolyl 4-hydroxylases as a promising approach to the therapy of cardiometabolic diseases.

Prolyl-4-hydroxylases of hypoxia-inducible factor (HIF-P4Hs) are enzymes that, under the conditions of normoxia, cause degradation of the HIF-transcriptional protein, which regulates a number of metabolic processes, including erythropoiesis, glucose level and lipid metabolism. In hypoxic conditions, on the contrary, their activity is suppressed and HIF stabilization takes place. This mechanism, i.e. stabilization of HIF by inhibition of HIF-P4Hs was the basis for the development of drugs designed for treatment of renal anemia, which are currently in stages 2 and 3 of clinical trials and are showing encouraging results. Recently, it has also been reported that inhibition of HIF-P4Hs can be effective in treatment of cardiometabolic diseases - coronary heart disease, hypertension, obesity, metabolic syndrome, diabetic cardiomyopathy and atherosclerosis. The review, based on the most recent data, discusses in detail molecular mechanisms of therapeutic effect of HIF-P4Hs inhibition in these pathological conditions and provides evidence that these mechanisms are associated with HIF stabilization and gene expression, improving perfusion and endothelial function, reprogramming metabolism from oxidative phosphorylation to anaerobic glycolysis, reducing inflammation and having beneficial effect on the innate immune system.

Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases.

The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications.

Protein ubiquitylation is a post-translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN-RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F-box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.