scholarly journals Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy

2018 ◽  
Author(s):  
Ieva Vasiliauskaité-Brooks ◽  
Robert D. Healey ◽  
Pascal Rochaix ◽  
Rémy Sounier ◽  
Claire Grison ◽  
...  
Keyword(s):  
Binding Site ◽  
Myeloid Leukemia ◽  
Transmembrane Domain ◽  
Domain Architecture ◽  
Computational Studies ◽  
Loss Of Function ◽  
Adiponectin Receptors ◽  
Structural Explanation ◽  
Type 3

AbstractAlkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

Identification of SAS4 and SAS5, Two Genes That Regulate Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Eugenia Y Xu ◽  
Susan Kim ◽  
Kirstin Replogle ◽  
Jasper Rine ◽  
David H Rivier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Myeloid Leukemia ◽  
Null Alleles ◽  
Human Genes ◽  
Complementation Groups ◽  
A Site ◽  
Acute Myeloid

Abstract In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the α-mating phenotype to MATα HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3470-3470
Author(s):  
Daniela Cilloni ◽  
Monica Pradotto ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Small Gtpases ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

2012 ◽  
Vol 303 (6) ◽  
pp. C673-C681 ◽  
Author(s):  
Rongbao Zhao ◽  
Daniel Sanghoon Shin ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Functional Role ◽  
Autosomal Recessive ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Autosomal Recessive Disorder ◽  
Binding Pocket ◽  
Loss Of Function ◽  
Substituted Cysteine Accessibility ◽  
Translocation Pathway

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.

Mir-590 Is a Novel STAT5 Regulated Oncogenic miRNA and Targets FasL In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3811-3811 ◽  
Author(s):  
Amanda J Favreau ◽  
Fariba Shaffiey ◽  
Erin Cross ◽  
Pradeep Sathyanarayana
Keyword(s):  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Functional Role ◽  
Luciferase Assay ◽  
Potential Binding ◽  
Flt3 Mutations ◽  
Acute Myeloid

Abstract The recent discovery of new molecular lesions with prognostic significance in acute myeloid leukemia (AML) is enhancing our understanding of leukemia biology and our ability to identify new therapeutic targets. Previously, using the unique leukemic myeloid progenitor line AML-193, we profiled IL-3-, GM-CSF-, and G-CSF-regulated miRNA signatures. 301 miRNAs were commonly regulated by these three cytokines, and the most highly induced miRNA was miR-590-5p. Herein, we have attempted to define the functional role and clinical relevance of miR-590 in AML. We first examined the relative miR-590 expression in steady state hematopoiesis and showed it was highest at CD34+ and declined its expression through myeloid lineage differentiation (ANOVA, p<0.0001). To functionally determine the role of increased miR-590 expression, we generated a gain-of-function model in human CD34+ hematopoietic stem cells (HSC) via lentivirus transduction. Increased expression of miR-590 in CD34+ cells resulted in significant increases in CFU-GM colonies, strongly suggesting that dysregulation of miR-590 expression may be myeloproliferative. In AML (n=33) and control (n=9) bone marrow samples, miR-590 expression was determined via RT-qPCR. miR-590-5p expression was highly upregulated in 22 of the samples (67%) compared to control subjects. In silico analysis of the miR-590-5p promoter revealed three potential binding sites for STAT5 (-249, -749, -1499). To functionally determine whether STAT5 directly regulates miR-590-5p expression, we performed a ChIP assay, which showed that STAT5 binds to the -749 region of miR-590-5p promoter. To conclusively determine the STAT5 binding sites, we cloned the miR-590 promoter in a luciferase vector and performed site directed mutagenesis for each potential binding site. This assay confirmed that the -749 binding site was the major STAT5 regulatory site for miR-590 (p<0.002). Importantly, constitutive activation of STAT5 is a hallmark of AML associated with FLT3 mutations, therefore, we set out to determine if specific STAT5 and FLT3 inhibitors could decrease miR-590 expression. We pretreated MV4-11 cells, which harbors the FLT-ITD mutation and has increased STAT5 activation, with 100uM STAT5 inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) for 90 minutes or 100nM FLT3 inhibitor (EMD Millipore, 343020) for 12 hours, both of which resulted in significant inhibition of miR-590-5p expression (p<0.05). To evaluate whether the AML samples with high miR-590 expression also possess elevated phospho-STAT5 or phospho-FLT3 levels, we performed immunohistochemistry analysis on a custom-made tissue microarray. In AML samples with high miR-590 levels, increased activation of FLT3 and STAT5 was observed compared to controls. Since FLT3 mutations result in decreased survival and poorer prognosis in AML, it may be that miR-590-5p plays an important role in the pathology of AML associated with dysregulated FLT3 and STAT5. To understand the complete functional role of miR-590 in AML, the predicted targets need to be identified and validated for their roles in leukemogenesis. Upon molecular screening of several predicted targets, FasL was experimentally found to be a conserved target of miR-590. More specifically, 3’UTR analysis of FasL revealed three potential seed sequences for miR-590 which have been verified experimentally via luciferase assay. Furthermore, significantly increased levels of FasL protein and transcript expression was detected in the MV4-11 cells stably expressing anti-miR-590 compared to control cells. Additionally, we identified the levels of Fas/CD95 (FasL receptor) on AML-193 and MV4-11 cell lines and found these cells had high Fas/CD95 expression on the cell surface as analyzed via flow cytometry. In order to determine the physiological significance of Fas/FasL, these cells were treated with soluble FasL (100ng) for 24 hours and apoptosis was analyzed via Annexin V staining. FasL treatment induced increased apoptosis compared to the untreated cells. Taken together, we have identified miR-590 as a candidate oncomiR that is regulated via the STAT5 pathway and targets FasL to promote cell survival. Thus, our data suggests that further understanding of miR-590’s role in AML may lead to development of novel anti-miR-590 therapeutic strategies in AML associated with dysregulated STAT5. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method

2013 ◽  
Vol 304 (12) ◽  
pp. C1159-C1167 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Rongbao Zhao ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Transmembrane Domain ◽  
Substrate Binding ◽  
Basolateral Membrane ◽  
Homology Model ◽  
Binding Pocket ◽  
Ependymal Cells ◽  
Loss Of Function ◽  
Substituted Cysteine Accessibility ◽  
Proximal Small Intestine

The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two loss-of-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146–167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the high-affinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe157, Gly158, and Leu161, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

scholarly journals Mutations in epigenetic modifiers in the pathogenesis and therapy of acute myeloid leukemia

Blood ◽  
2013 ◽  
Vol 121 (18) ◽  
pp. 3563-3572 ◽  
Author(s):  
Omar Abdel-Wahab ◽  
Ross L. Levine
Keyword(s):  
Acute Myeloid Leukemia ◽  
Posttranslational Modifications ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Genetic Alterations ◽  
Loss Of Function ◽  
Epigenetic Modifiers ◽  
Genetic Abnormalities ◽  
Acute Myeloid

Abstract Recent studies of the spectrum of somatic genetic alterations in acute myeloid leukemia (AML) have identified frequent somatic mutations in genes that encode proteins important in the epigenetic regulation of gene transcription. This includes proteins involved in the modification of DNA cytosine residues and enzymes which catalyze posttranslational modifications of histones. Here we describe the clinical, biological, and therapeutic relevance of mutations in epigenetic regulators in AML. In particular, we focus on the role of loss-of-function mutations in TET2, gain-of-function mutations in IDH1 and IDH2, and loss-of-function mutations in ASXL1 and mutations of unclear impact in DNMT3A in AML pathogenesis and therapy. Multiple studies have consistently identified that mutations in these genes have prognostic relevance, particularly in intermediate-risk AML patients, arguing for inclusion of mutational testing of these genetic abnormalities in routine clinical practice. Moreover, biochemical, biological, and epigenomic analyses of the effects of these mutations have informed the development of novel therapies which target pathways deregulated by these mutations. Our understanding of the effects of these mutations on hematopoiesis and potential for therapeutic targeting of specific AML subsets is also reviewed here.

Epigenetic Suppression of the CTNNA1 Gene, Encoding the α-Catenin Protein, which Is Located in the 5q31 Critical Deleted Region in Malignant Myeloid Disorders with del(5q).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 203-203
Author(s):  
Ting Xi Liu ◽  
Michael Becker ◽  
Karl Hsu ◽  
Jaroslav Jelinek ◽  
Min Deng ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Genes Expression ◽  
Polymerase Chain ◽  
Epigenetic Suppression

Abstract Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Although the affected genes have not been identified, two critical deleted regions (CDR) on 5q have been established, a proximal CDR located in 5q31, and another distal CDR in 5q32–33. We investigated the expression of 28 genes located in the 5q31 CDR, in enriched populations of normal hematopoietic stem cells (HSCs) and leukemic initiating cells (L-ICs) by the reverse transcription polymerase chain reaction (RT-PCR). Of the 28 5q CDR genes investigated, 12 (42%) were expressed in HSCs (CD34+Thy-1+CD38-Lin-), including HNRPA0 (Hs.77492), LOC51306 (Hs.82035), SMAP (Hs.5464), CDC23 (Hs.153546), LOC51307 (Hs.102469), LOC51780 (Hs.24125), EGRI (Hs.326035), ETF1 (Hs.77324), HSBPA9B (Hs.3069), CTNNA1 (Hs.178452), MATR3 (Hs.223745) and UBE2D2 (Hs.108332). We also isolated L-ICs (CD34+CD123+CD38-Lin-) from 11 cases of MDS/AML with 5q-, and investigated their expression levels of these 12 HSC genes. Expression of CTNNA1 was not detected in the L-ICs of 8 of the 11 cases of del(5q) MDS/AML using standard RT-PCR, while the other 11 genes were expressed in each patient at levels comparable to those of HSCs. Quantitative analysis of CTNNA1 expression in L-ICs by real-time RT-PCR confirmed expression levels <30% of HSC levels in 4 of the 6 cases tested with del(5q) MDS/AML. Analysis of protein expression by immunofluorescence also verified a lack of detectable CTNNA1 in L-ICs for the two del(5q) cases tested. CTNNA1 expression levels were normal in each of the 11 non-del(5q) MDS/AML cases tested. Methylation of the CTNNA1 promoter region correlated with the downregulation of CTNNA1 transcript in the del(5q) HL-60 cell line, in which CTNNA1 expression was partially restored following treatment with 5-aza-2-deoxycytidine. Our results suggest that epigenetic suppression of the expression of the intact CTNNA1 allele may contribute to the pathogenesis of MDS/AML with del(5q). We are currently using mouse and zebrafish models to study the role of CTNNA1 loss-of-function in del(5q) MDS/AML leukemogenesis.

scholarly journals Combined loss of function of two different loci of miR-15/16 drives the pathogenesis of acute myeloid leukemia

2020 ◽  
Vol 117 (22) ◽  
pp. 12332-12340 ◽  
Author(s):  
Francesca Lovat ◽  
Giovanni Nigita ◽  
Rosario Distefano ◽  
Tatsuya Nakamura ◽  
Pierluigi Gasparini ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Combination Therapies ◽  
Loss Of Function ◽  
Double Knockout ◽  
Incurable Disease ◽  
Acute Myeloid

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS–AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS–AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.

scholarly journals Recurrent SETD2 mutation in NPM1-mutated acute myeloid leukemia

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Jiewen Sun ◽  
Wenjuan Yu ◽  
Xiang Zhang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tumor Suppressor ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Functional Study ◽  
Loss Of Function ◽  
Direct Target ◽  
And Function ◽  
Acute Myeloid

Abstract SETD2 is the only methyltransferase for H3K36me3, and our previous study has firstly demonstrated that it functioned as one tumor suppressor in hematopoiesis. Consistent with it, SETD2 mutation, which led to its loss of function, was identified in AML. However, the distribution and function of SETD2 mutation in AML remained largely unknown. Herein, we integrated SETD2-mutated AML cases from our center and literature reports, and found that NPM1 mutation was the most common concomitant genetic alteration with SETD2 mutation in AML, with its frequency even higher than MLL rearrangement and AML1-ETO. Though this result indicated the cooperation of SETD2 and NPM1 mutations in leukemogenesis, our functional study showed that SETD2 was required for the proliferation of NPM1-mutated AML cell line OCI-AML3, but not MLL-rearranged AML cell line THP-1, via maintaining its direct target NPM1 expression, which was just opposite to its role of tumor suppressor. Therefore, we speculated that SETD2 possibly had two different faces in distinct subtypes and stages of AML.

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