| 1. |
- Adhikari, Deepak, et al.
(author)
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Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes
- 2012
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 21:11, s. 2476-2484
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Journal article (peer-reviewed)abstract
- Mammalian oocytes are arrested at the prophase of meiosis I during fetal or postnatal development, and the meiosis is resumed by the preovulatory surge of luteinizing hormone. The in vivo functional roles of cyclin-dependent kinases (Cdks) during the resumption of meiosis in mammalian oocytes are largely unknown. Previous studies have shown that deletions of Cdk3, Cdk4 or Cdk6 in mice result in viable animals with normal oocyte maturation, indicating that these Cdks are not essential for the meiotic maturation of oocytes. In addition, conventional knockout of Cdk1 and Cdk2 leads to embryonic lethality and postnatal follicular depletion, respectively, making it impossible to study the functions of Cdk1 and Cdk2 in oocyte meiosis. In this study, we generated conditional knockout mice with oocyte-specific deletions of Cdk1 and Cdk2. We showed that the lack of Cdk1, but not of Cdk2, leads to female infertility due to a failure of the resumption of meiosis in the oocyte. Re-introduction of Cdk1 mRNA into Cdk1-null oocytes largely resumed meiosis. Thus, Cdk1 is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. We also found that Cdk1 maintains the phosphorylation status of protein phosphatase 1 and lamin A/C in oocytes in order for meiosis resumption to occur.
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| 2. |
- Adhikari, Deepak, et al.
(author)
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Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan
- 2016
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In: Cell Research. - : Springer Science and Business Media LLC. - 1001-0602 .- 1748-7838. ; 26, s. 1212-1225
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Journal article (peer-reviewed)abstract
- © 2016 IBCB, SIBS, CAS. A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1 T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals.
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| 3. |
- Adhikari, Deepak, et al.
(author)
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Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II.
- 2014
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In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 206:7, s. 843-853
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Journal article (peer-reviewed)abstract
- In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.
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| 4. |
- Bisteau, Xavier, et al.
(author)
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The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis
- 2020
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In: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594.
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Journal article (peer-reviewed)abstract
- Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.
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| 5. |
- Caldez, Matias J, et al.
(author)
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Cell cycle regulation in NAFLD: when imbalanced metabolism limits cell division : when imbalanced metabolism limits cell division
- 2020
-
In: Hepatology international. - : Springer Science and Business Media LLC. - 1936-0533 .- 1936-0541. ; 14:4, s. 463-474
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Research review (peer-reviewed)abstract
- Cell division is essential for organismal growth and tissue homeostasis. It is exceptionally significant in tissues chronically exposed to intrinsic and external damage, like the liver. After decades of studying the regulation of cell cycle by extracellular signals, there are still gaps in our knowledge on how these two interact with metabolic pathways in vivo. Studying the cross-talk of these pathways has direct clinical implications as defects in cell division, signaling pathways, and metabolic homeostasis are frequently observed in liver diseases. In this review, we will focus on recent reports which describe various functions of cell cycle regulators in hepatic homeostasis. We will describe the interplay between the cell cycle and metabolism during liver regeneration after acute and chronic damage. We will focus our attention on non-alcoholic fatty liver disease, especially non-alcoholic steatohepatitis. The global incidence of non-alcoholic fatty liver disease is increasing exponentially. Therefore, understanding the interplay between cell cycle regulators and metabolism may lead to the discovery of novel therapeutic targets amenable to intervention.
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| 6. |
- Colon-Mesa, Ignacio, et al.
(author)
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p27Kip1 Deficiency Impairs Brown Adipose Tissue Function Favouring Fat Accumulation in Mice
- 2023
-
In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:3
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
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| 8. |
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| 9. |
- Kaldis, Philipp, et al.
(author)
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Molecular basis of the reaction mechanism of the methyltransferase HENMT1
- 2024
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In: PLoS ONE. - 1932-6203. ; 19:1
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Keles, Umur, et al.
(author)
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Liver-derived metabolites as signaling molecules in fatty liver disease
- 2023
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In: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 80:1, s. 1-14
-
Research review (peer-reviewed)abstract
- 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.
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| 11. |
- Na Zhao, Li, et al.
(author)
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Cascading proton transfers are a hallmark of the catalytic mechanism of SAM-dependent methyltransferases
- 2020
-
In: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 594:13, s. 2128-2139
-
Journal article (peer-reviewed)abstract
- The S-adenosyl-L-methionine (SAM)-dependent methyltransferases attach a methyl group to the deprotonated methyl lysine (Kme0) using SAM as a donor. An intriguing, yet unanswered, question is how the deprotonation of the methyl lysine takes place which results in a lone pair of electrons at the Nϵ atom of the methyl lysine for the following methyl transfer. PRDM9, one of the few methyltransferases with well-defined enzyme activity in vitro and in vivo, is a good representative of the PR/SET domain methyltransferase family to study the deprotonation and subsequently the methyl transfer. The reaction consists of two progressing steps: (i) the absolutely required substrate methyl lysine deprotonation and (ii) the transfer of the methyl group to the deprontonated methyl lysine. We use empirical valence bond (EVB) simulations to evaluate Y357 at the active site as potential general base for the deprotonation of the methyl lysine. Indeed, our study has found that the pKa of Tyr357 is low enough to make it an ideal candidate for proton abstraction from the methyl lysine. The partially deprontonated Tyr357 is able to change its H-bond pattern thus bridging two proton tunneling states (OH- H 0-Tyr357 and Kme0-Nϵ H O-Tyr357) and providing a cascading proton transfer from Tyr357 to hydroxide, generating deprotonated Tyr357 and then from Kme0 to the deprotonated Tyr357 resulting in deprotonated methyl lysine. This cascading proton transfer shortens the lifespan of the labile intermediates, and affects the conformational changes during the product release important to promote the proton release to the bulk solvent. Our computational efforts have uncovered a new catalytic mechanism to unravel the unanswered question about the deprotonation of the methyl lysine in methyltransferases.
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| 12. |
- Narayanaswamy, Pradeep, et al.
(author)
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MetaboKit : a comprehensive data extraction tool for untargeted metabolomics
- 2020
-
In: Molecular omics. - : Royal Society of Chemistry (RSC). - 2515-4184. ; 16:5, s. 436-447
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Journal article (peer-reviewed)abstract
- We have developed MetaboKit, a comprehensive software package for compound identification and relative quantification in mass spectrometry-based untargeted metabolomics analysis. In data dependent acquisition (DDA) analysis, MetaboKit constructs a customized spectral library with compound identities from reference spectral libraries, adducts, dimers, in-source fragments (ISF), MS/MS fragmentation spectra, and more importantly the retention time information unique to the chromatography system used in the experiment. Using the customized library, the software performs targeted peak integration for precursor ions in DDA analysis and for precursor and product ions in data independent acquisition (DIA) analysis. With its stringent identification algorithm requiring matches by both MS and MS/MS data, MetaboKit provides identification results with significantly greater specificity than the competing software packages without loss in sensitivity. The proposed MS/MS-based screening of ISFs also reduces the chance of unverifiable identification of ISFs considerably. MetaboKit's quantification module produced peak area values highly correlated with known concentrations in a DIA analysis of the metabolite standards at both MS1 and MS2 levels. Moreover, the analysis of Cdk1Liv-/- mouse livers showed that MetaboKit can identify a wide range of lipid species and their ISFs, and quantitatively reconstitute the well-characterized fatty liver phenotype in these mice. In DIA data, the MS1-level and MS2-level peak area data produced similar fold change estimates in the differential abundance analysis, and the MS2-level peak area data allowed for quantitative comparisons in compounds whose precursor ion chromatogram was too noisy for peak integration.
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| 13. |
- Niska-Blakie, Joanna, et al.
(author)
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Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype
- 2019
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In: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071.
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Journal article (peer-reviewed)abstract
- SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as “crown-like” structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.
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| 14. |
- Ow, Jin Rong, et al.
(author)
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Remodelling of whole-body lipid metabolism and a diabetic-like phenotype caused by loss of CDK1 and hepatocyte division
- 2020
-
In: eLife. - 2050-084X. ; 9
-
Journal article (peer-reviewed)abstract
- Cell cycle progression and lipid metabolism are well-coordinated processes required for proper cell proliferation. In liver diseases that arise from dysregulated lipid metabolism, proliferation is diminished. To study the outcome of CDK1 loss and blocked hepatocyte proliferation on lipid metabolism and the consequent impact on whole-body physiology, we performed lipidomics, metabolomics, and RNA-seq analyses on a mouse model. We observed reduced triacylglycerides in liver of young mice, caused by oxidative stress that activated FOXO1 to promote expression of ATGL. Additionally, we discovered that hepatocytes displayed malfunctioning b-oxidation, reflected by increased acylcarnitines and reduced b-hydroxybutyrate. This led to elevated plasma free fatty acids, which were transported to the adipose tissue for storage and triggered greater insulin secretion. Upon aging, chronic hyperinsulinemia resulted in insulin resistance and hepatic steatosis through activation of LXR. Here we demonstrate that loss of hepatocyte proliferation is not only an outcome but possibly causative for liver pathology.
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| 15. |
- Palmer, Nathan, et al.
(author)
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A novel function for CDK2 activity at meiotic crossover sites
- 2020
-
In: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 18:10
-
Journal article (peer-reviewed)abstract
- Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.
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| 16. |
- Palmer, Nathan, et al.
(author)
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Diverse roles for CDK-associated activity during spermatogenesis
- 2019
-
In: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 593:20, s. 2925-2949
-
Research review (peer-reviewed)abstract
- The primary function of cyclin-dependent kinases (CDKs) in complex with their activating cyclin partners, is to promote mitotic division in somatic cells. This canonical cell cycle-associated activity is also crucial for fertility as it allows the proliferation and differentiation of stem cells within the reproductive organs to generate meiotically competent cells. Intriguingly several CDKs exhibit meiosis-specific functions and are essential for the completion of the two reductional meiotic divisions required to generate haploid gametes. These meiosis-specific functions are mediated by both known CDK/cyclin complexes and meiosis-specific CDK-regulators and are important for a variety of processes during meiotic prophase. The majority of meiotic defects observed upon deletion of these proteins occur during the extended prophase I of the first meiotic division. Importantly a lack of redundancy is seen within the meiotic arrest phenotypes described for many of these proteins suggesting intricate layers of cell cycle control are required for normal meiotic progression. Using the process of male germ cell development (spermatogenesis) as a reference, this review seeks to highlight the diverse roles of selected CDKs their activators, and their regulators during gametogenesis.
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| 17. |
- Palmer, Nathan, et al.
(author)
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Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity
- 2023
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:3
-
Journal article (peer-reviewed)abstract
- 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.
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| 18. |
- Palmer, Nathan, et al.
(author)
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Less-well known functions of cyclin/CDK complexes
- 2020
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In: Seminars in Cell & Developmental Biology. - : Elsevier BV. - 1084-9521. ; 107, s. 54-62
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Research review (peer-reviewed)abstract
- Cyclin-dependent kinases (CDKs) are activated by cyclins, which play important roles in dictating the actions of CDK/cyclin complexes. Cyclin binding influences the substrate specificity of these complexes in addition to their susceptibility to inhibition or degradation. CDK/cyclin complexes are best known to promote cell cycle progression in the mitotic cell cycle but are also crucial for important cellular processes not strictly associated with cellular division. This chapter primarily explores the understudied topic of CDK/cyclin complex functionality during the DNA damage response. We detail how CDK/cyclin complexes perform dual roles both as targets of DNA damage checkpoint signaling as well as effectors of DNA repair. Additionally, we discuss the potential CDK-independent roles of cyclins in these processes and the impact of such roles in human diseases such as cancer. Our goal is to place the spotlight on these important functions of cyclins either acting as independent entities or within CDK/cyclin complexes which have attracted less attention in the past. We consider that this will be important for a more complete understanding of the intricate functions of cell cycle proteins in the DNA damage response.
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| 19. |
- Rajareddy, Singareddy, et al.
(author)
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p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice
- 2007
-
In: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 21:9, s. 2189-2202
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Journal article (peer-reviewed)abstract
- In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27kip1 or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27−/−) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27+/+ mice. Moreover, in p27−/− ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27−/− ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27−/− ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.
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| 20. |
- Sanches, Jose Marcos, et al.
(author)
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Pathophysiology of type 2 diabetes and the impact of altered metabolic interorgan crosstalk
- 2023
-
In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 290:3, s. 620-648
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Research review (peer-reviewed)abstract
- 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 β-cell compensatory mechanisms overcoming peripherally developed insulin resistance is a paramount factor leading to disturbed blood glucose levels and lipid metabolism. Impaired β-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.
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| 21. |
- Tanaka, Tomoyuki, et al.
(author)
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Regulation of osteoblast to osteocyte differentiation by cyclin-dependent kinase-1
- 2023
-
In: Advanced biology. - 2701-0198. ; 7:12
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Journal article (peer-reviewed)abstract
- 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-Cdk1 KO ) 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-Cdk1 KO mice. Four weeks of Parathyroid hormone administration partially recovers the trabecular bone loss in Dmp1-Cdk1 KO 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.
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| 22. |
- Xu, Jianliang, et al.
(author)
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Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis
- 2021
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In: Gastroenterology. - : Elsevier BV. - 1528-0012 .- 0016-5085. ; 160:6, s. 2103-2118
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Journal article (peer-reviewed)abstract
- BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Since the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2.METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver and bile and .by EM, histology and immunostaining. TJ barrier permeability was evaluated using FITC-Dextran (4kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury.RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density and aberrant Radixin and BSEP distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid (BA) transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA-diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. TCPOBOP ameliorated CA-induced injury by enhancing Cyp2b10 expression and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes only showed mild CA-induced liver injury.CONCLUSION: Tjp2 is required for normal cortical distribution of Radixin, canalicular volume regulation and microvilli density, Its inactivation deregulated expression of Cldn1 and key BA transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.
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| 23. |
- Zhao, Lina, et al.
(author)
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Histidine protonation states are key in the LigI catalytic reaction mechanism
- 2022
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In: Proteins. - : Wiley. - 0887-3585. ; 90:1, s. 123-130
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Journal article (peer-reviewed)abstract
- Lignin is one of the world's most abundant organic polymers, and 2-pyrone-4,6-dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the &ip.eop; site, however, protonation at the δ site may be favored during specific steps of the reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.
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| 24. |
- Zhao, Li Na, et al.
(author)
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Pairing structural reconstruction with catalytic competence to evaluate the mechanisms of key enzymes in the folate-mediated one-carbon pathway
- 2023
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 290:9, s. 2279-2291
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Research review (peer-reviewed)abstract
- 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, which are required for nucleotide synthesis, methylation and metabolism, 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 in their active form are not available, we used homology modeling integrated with the interpretation of the reaction mechanism, and have reconstructed the most likely scenario for the reactions to take place paired with their catalytic cycle that provides a testable framework for this pathway.
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| 25. |
- Zhao, Li-Na, et al.
(author)
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Proof-of-concept method to study uncharacterized methyltransferases using PRDM15
- 2023
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 24:2, s. 1-12
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Journal article (peer-reviewed)abstract
- 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.
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| 26. |
- Zhao, Li Na, et al.
(author)
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The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)
- 2022
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In: PLoS Computational Biology. - : Public Library of Science (PLoS). - 1553-7358. ; 18:5
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Journal article (peer-reviewed)abstract
- Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate Mg(A)2+, while Mg(A)2+ together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate Mg(B)2+, and Mg(B)2+ further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of Mg(A)2+, Mg(B)2+ and Pi. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.
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| 27. |
- Zhao, Li Na, et al.
(author)
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Therapeutic targeting of the mitochondrial one-carbon pathway: perspectives, pitfalls, and potential
- 2021
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In: Oncogene. - : Springer Science and Business Media LLC. - 1476-5594 .- 0950-9232.
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Research review (peer-reviewed)abstract
- Most of the drugs currently prescribed for cancer treatment are riddled with substantial side effects. In order to develop more effective and specific strategies to treat cancer, it is of importance to understand the biology of drug targets, particularly the newly emerging ones. A comprehensive evaluation of these targets will benefit drug development with increased likelihood for success in clinical trials. The folate-mediated one-carbon (1C) metabolism pathway has drawn renewed attention as it is often hyperactivated in cancer and inhibition of this pathway displays promise in developing anticancer treatment with fewer side effects. Here, we systematically review individual enzymes in the 1C pathway and their compartmentalization to mitochondria and cytosol. Based on these insight, we conclude that (1) except the known 1C targets (DHFR, GART, and TYMS), MTHFD2 emerges as good drug target, especially for treating hematopoietic cancers such as CLL, AML, and T-cell lymphoma; (2) SHMT2 and MTHFD1L are potential drug targets; and (3) MTHFD2L and ALDH1L2 should not be considered as drug targets. We highlight MTHFD2 as an excellent therapeutic target and SHMT2 as a complementary target based on structural/biochemical considerations and up-to-date inhibitor development, which underscores the perspectives of their therapeutic potential.
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