Regulation of biological processes by ubiquitin ligases: a focus on the Pagano Lab's contribution.

Protein homeostasis depends on many fundamental processes including mRNA synthesis, translation, post-translational modifications, and proteolysis. In the late 70s and early 80s the discovery that the small 76 amino acid protein ubiquitin could be attached to target proteins via a multi-stage process involving ubiquitin-activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases, revealed an exciting new post-translational mechanism to regulate protein degradation. This cellular system was uncovered using biochemical methods by Avram Hershko, who would later won the Nobel prize for this discovery; however, the biological functions of ubiquitin ligases remained unknown for many years. It was initially described that ubiquitin modifies proteins at one or more lysine residues and once a long ubiquitin chain was assembled, proteins were degraded by the proteasome. Now we know that proteins can be mono-, multimono-, homotypic poly-, or heterotypic poly-ubiquitylated, each of which confers a specific signal that goes beyond protein degradation regulating additional key cellular functions such as signal transduction, protein localization, recognition of damaged proteins, etc.

Molecular basis of the reaction mechanism of the methyltransferase HENMT1.

PIWI-interacting RNAs (piRNAs) are important for ensuring the integrity of the germline. 3'-terminal 2'-O-methylation is essential for piRNA maturation and to protect them from degradation. HENMT1 (HEN Methyltransferase 1) carries out the 2'-O-methylation, which is of key importance for piRNA stability and functionality. However, neither the structure nor the catalytic mechanism of mammalian HENMT1 have been studied. We have constructed a catalytic-competent HENMT1 complex using computational approaches, in which Mg2+ is primarily coordinated by four evolutionary conserved residues, and is further auxiliary coordinated by the 3'-O and 2'-O on the 3'-terminal nucleotide of the piRNA. Our study suggests that metal has limited effects on substrate and cofactor binding but is essential for catalysis. The reaction consists of deprotonation of the 2'-OH to 2'-O and a methyl transfer from SAM to the 2'-O. The methyl transfer is spontaneous and fast. Our in-depth analysis suggests that the 2'-OH may be deprotonated before entering the active site or it may be partially deprotonated at the active site by His800 and Asp859, which are in a special alignment that facilitates the proton transfer out of the active site. Furthermore, we have developed a detailed potential reaction scenario indicating that HENMT1 is Mg2+ utilizing but is not a Mg2+ dependent enzyme.

Regulation of Osteoblast to Osteocyte Differentiation by Cyclin-Dependent Kinase-1.

Osteocytes have recently been identified as a new regulator of bone remodeling, but the detailed mechanism of their differentiation from osteoblasts remains unclear. The purpose of this study is to identify cell cycle regulators involved in the differentiation of osteoblasts into osteocytes and determine their physiological significance. The study uses IDG-SW3 cells as a model for the differentiation from osteoblasts to osteocytes. Among the major cyclin-dependent kinases (Cdks), Cdk1 is most abundantly expressed in IDG-SW3 cells, and its expression is down-regulated during differentiation into osteocytes. Inhibition of CDK1 activity reduces IDG-SW3 cell proliferation and differentiation into osteocytes. Osteocyte and Osteoblast-specific Cdk1 knockout in mice (Dmp1-Cdk1KO ) results in trabecular bone loss. Pthlh expression increases during differentiation, but inhibiting CDK1 activity reduces Pthlh expression. Parathyroid hormone-related protein concentration is reduced in the bone marrow of Dmp1-Cdk1KO mice. Four weeks of Parathyroid hormone administration partially recovers the trabecular bone loss in Dmp1-Cdk1KO mice. These results demonstrate that Cdk1 plays an essential role in the differentiation from osteoblast to osteocyte and the acquisition and maintenance of bone mass. The findings contribute to a better understanding of the mechanisms of bone mass regulation and can help develop efficient therapeutic strategies for osteoporosis treatment.

Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity.

Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.

p27Kip1 Deficiency Impairs Brown Adipose Tissue Function Favouring Fat Accumulation in Mice.

The aim of this work was to investigate the effect of the whole-body deletion of p27 on the activity of brown adipose tissue and the susceptibility to develop obesity and glucose homeostasis disturbances in mice, especially when subjected to a high fat diet. p27 knockout (p27-/-) and wild type (WT) mice were fed a normal chow diet or a high fat diet (HFD) for 10-weeks. Body weight and composition were assessed. Insulin and glucose tolerance tests and indirect calorimetry assays were performed. Histological analysis of interscapular BAT (iBAT) was carried out, and expression of key genes/proteins involved in BAT function were characterized by qPCR and Western blot. iBAT activity was estimated by 18F-fluorodeoxyglucose (18FDG) uptake with microPET. p27-/- mice were more prone to develop obesity and insulin resistance, exhibiting increased size of all fat depots. p27-/- mice displayed a higher respiratory exchange ratio. iBAT presented larger adipocytes in p27-/- HFD mice, accompanied by downregulation of both Glut1 and uncoupling protein 1 (UCP1) in parallel with defective insulin signalling. Moreover, p27-/- HFD mice exhibited impaired response to cold exposure, characterized by a reduced iBAT 18FDG uptake and difficulty to maintain body temperature when exposed to cold compared to WT HFD mice, suggesting reduced thermogenic capacity. These data suggest that p27 could play a role in BAT activation and in the susceptibility to develop obesity and insulin resistance.

Proof-of-Concept Method to Study Uncharacterized Methyltransferases Using PRDM15.

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15.

Pairing structural reconstruction with catalytic competence to evaluate the mechanisms of key enzymes in the folate-mediated one-carbon pathway.

Mammalian metabolism comprises a series of interlinking pathways that include two major cycles: the folate and methionine cycles. The folate-mediated metabolic cycle uses several oxidation states of tetrahydrofolate to carry activated one-carbon units to be readily used and interconverted within the cell. They are required for nucleotide synthesis, methylation and metabolism, and particularly for proliferation of cancer cells. Based on the latest progress in genome-wide CRISPR loss-of-function viability screening of 789 cell lines, we focus on the most cancer-dependent enzymes in this pathway, especially those that are hyperactivated in cancer, to provide new insight into the chemical basis for cancer drug development. Since the complete 3D structure of several of these enzymes of the one-carbon pathway in their active form are not available, we used homology modelling integrated with the interpretation of the reaction mechanism. In addition, have reconstructed the most likely scenario for the reactions taking place paired with their catalytic competence that provides a testable framework for this pathway.

Pathophysiology of type 2 diabetes and the impact of altered metabolic interorgan crosstalk.

Diabetes is a complex and multifactorial disease that affects millions of people worldwide, reducing the quality of life significantly, and results in grave consequences for our health care system. In type 2 diabetes (T2D), the lack of ß-cell compensatory mechanisms overcoming peripherally developed insulin resistance is a paramount factor leading to disturbed blood glucose levels and lipid metabolism. Impaired ß-cell functions and insulin resistance have been studied extensively resulting in a good understanding of these pathways but much less is known about interorgan crosstalk, which we define as signaling between tissues by secreted factors. Besides hormones and organokines, dysregulated blood glucose and long-lasting hyperglycemia in T2D is associated with changes in metabolism with metabolites from different tissues contributing to the development of this disease. Recent data suggest that metabolites, such as lipids including free fatty acids and amino acids, play important roles in the interorgan crosstalk during the development of T2D. In general, metabolic remodeling affects physiological homeostasis and impacts the development of T2D. Hence, we highlight the importance of metabolic interorgan crosstalk in this review to gain enhanced knowledge of the pathophysiology of T2D, which may lead to new therapeutic approaches to treat this disease.

Liver-derived metabolites as signaling molecules in fatty liver disease.

Excessive fat accumulation in the liver has become a major health threat worldwide. Unresolved fat deposition in the liver can go undetected until it develops into fatty liver disease, followed by steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Lipid deposition in the liver is governed by complex communication, primarily between metabolic organs. This can be mediated by hormones, organokines, and also, as has been more recently discovered, metabolites. Although how metabolites from peripheral organs affect the liver is well documented, the effect of metabolic players released from the liver during the development of fatty liver disease or associated comorbidities needs further attention. Here we focus on interorgan crosstalk based on metabolites released from the liver and how these molecules act as signaling molecules in peripheral tissues. Due to the liver's specific role, we are covering lipid and bile mechanism-derived metabolites. We also discuss the high sucrose intake associated with uric acid release from the liver. Excessive fat deposition in the liver during fatty liver disease development reflects disrupted metabolic processes. As a response, the liver secretes a variety of signaling molecules as well as metabolites which act as a footprint of the metabolic disruption. In the coming years, the reciprocal exchange of metabolites between the liver and other metabolic organs will gain further importance and will help to better understand the development of fatty liver disease and associated diseases.