Molecular Dynamics Simulation Studies of GLUT4: Substrate-Free and Substrate-Induced Dynamics and ATP-Mediated Glucose Transport Inhibition

BACKGROUND: Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. CONCLUSIONS/SIGNIFICANCE: In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational changes in GLUT4 induced by substrate and its regulator

Error-corrected sequencing strategies enable comprehensive detection of leukemic mutations relevant for diagnosis and minimal residual disease monitoring

BACKGROUND: Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. METHODS: Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4-11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. RESULTS: Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. CONCLUSIONS: Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking

A case-control analysis of common variants in GIP with type 2 diabetes and related biochemical parameters in a South Indian population

<p>Abstract</p> <p>Background</p> <p>Glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, which plays a crucial role in the secretion of insulin upon food stimulus and in the regulation of postprandial glucose level. It also exerts an effect on the synthesis and secretion of lipoprotein lipase, from adipocytes, important for lipid metabolism. The aim of our study was to do a case-control association analysis of common variants in <it>GIP </it>in association with type 2 diabetes and related biochemical parameters.</p> <p>Method</p> <p>A total of 2000 subjects which includes 1000 (584M/416F) cases with type 2 diabetes and 1000 (470M/530F) normoglycemic control subjects belonging to Dravidian ethnicity from South India were recruited to assess the effect of single nucleotide polymorphisms (SNPs) in <it>GIP </it>(rs2291725, rs2291726, rs937301) on type 2 diabetes in a case-control manner. The SNPs were genotyped by using tetra primer amplification refractory mutation system-PCR (ARMS PCR). For statistical analysis, our study population was divided into sub-groups based on gender (male and female). Association analysis was carried out using chi-squared test and the comparison of biochemical parameters among the three genotypes were performed using analysis of covariance (ANCOVA).</p> <p>Result</p> <p>Initial analysis revealed that, out of the total three SNPs selected for the present study, two SNPs namely rs2291726 and rs937301 were in complete linkage disequilibrium (LD) with each other. Therefore, only two SNPs, rs2291725 and rs2291726, were genotyped for the association studies. No significant difference in the allele frequency and genotype distribution of any of the SNPs in <it>GIP </it>were observed between cases and controls (<it>P </it>> 0.05). Analysis of biochemical parameters among the three genotypes showed a significant association of total cholesterol (<it>P </it>= 0.042) and low density lipoprotein (LDL) with the G allele of the SNP rs2291726 in <it>GIP </it>(<it>P </it>= 0.004), but this was observed only in the case of female subjects. However this association does not remain significant after correction for multiple testing by Bonferroni's inequality method.</p> <p>Conclusion</p> <p>No statistically significant association was observed between any of the SNPs analysed and type 2 diabetes in our population. But the analysis of biochemical parameters indicates that the G allele in rs2291726 may be a putative risk allele for increased LDL cholesterol and further studies in other population needs to be carried out for ascertaining its role in cholesterol metabolism and subsequent cardiovascular risk.</p

Case-Control Analysis of SNPs in GLUT4, RBP4 and STRA6: Association of SNPs in STRA6 with Type 2 Diabetes in a South Indian Population

BACKGROUND: The inverse relationship between GLUT4 and RBP4 expression is known to play a role in the pathogenesis of type 2 diabetes. Elevated levels of RBP4 were shown to cause insulin resistance in muscles and liver. Identification of STRA6 as a cell surface receptor for RBP4 provides further link in this axis and hence we analyzed SNPs in these three genes for association with type 2 diabetes in a South Indian population. METHODOLOGY/PRINCIPAL FINDINGS: Selected SNPs in the three genes were analyzed in a total of 2002 individuals belonging to Dravidian ethnicity, South India, by Tetra Primer ARMS PCR or RFLP PCR. Allele frequencies and genotype distribution were calculated in cases and controls and were analyzed for association by Chi-squared test and Logistic regression. Haplotype analysis was carried out for each gene by including all the markers in a single block. We observed a significant association of three SNPs, rs974456, rs736118, and rs4886578 in STRA6 with type 2 diabetes (P = 0.001, OR 0.79[0.69-0.91], P = 0.003, OR 0.81[0.71-0.93], and P = 0.001, OR 0.74[0.62-0.89] respectively). None of the SNPs in RBP4 and GLUT4 showed any association with type 2 diabetes. Haplotype analysis revealed that two common haplotypes H1 (111, P = 0.001, OR 1.23[1.08-1.40]) and H2 (222, P = 0.002 OR 0.73[0.59-0.89]) in STRA6, H6 (2121, P = 0.006, OR 1.69[1.51-2.48]) in RBP4 and H4 (2121, P = 0.01 OR 1.41[1.07-1.85]) in GLUT4 were associated with type 2 diabetes. CONCLUSION: SNPs in STRA6, gene coding the cell surface receptor for RBP4, were significantly associated with type 2 diabetes and further genetic and functional studies are required to understand and ascertain its role in the manifestation of type 2 diabetes

Abstract 5870: Epigenetic drug treatment overcomes osteoblast-induced chemoprotection by suppressing cell adhesion and related signaling

Abstract Acute lymphoblastic leukemia (ALL) is the most commonly diagnosed cancer in pediatric patients. Recent advances have allowed for improved efficacy of treatment allowing 95% of patients to achieve remission following chemotherapy. However, nearly 20% will have a second recurrence that is often more aggressive and difficult to treat. One reason for this high rate of relapse may be due to the role of the bone marrow microenvironment (BM) consisting of osteoblasts, endothelial cells, adipocytes, and stromal cells as well as extracellular matrix proteins. Interaction between leukemic cells and BM elements activates intracellular signaling pathways that protect ALL cells from chemotherapy. Many of these pathways can be aberrantly activated due to changes in methylation patterns during leukemogenesis. Therefore, utilizing epigenetic modifiers offers a unique approach to overcoming the chemoprotective effects of the BM. The aim of our study is to identify if the combination of the epigenetic drugs azacitidine (DNA methyltransferase inhibitor) and panobinostat (histone deacetylase inhibitor) is successful in overcoming these effects. We demonstrated that azacitidine and panobinostat are more effective in killing ALL cells in coculture with osteoblasts than chemotherapy alone. This required direct interaction between ALL cells and osteoblasts and could not be replicated when ALL cells were suspended in Transwells above the osteoblast monolayer. Additionally, ALL cells pretreated with non-killing concentrations of azacitidine and panobinostat were sensitized to chemotherapy even in the presence of osteoblasts. These effects were replicated ex vivo in primary ALL patient samples with a variety of cytogenetic characteristics. These patient samples have been xenografted in mice to observe the efficacy of this combination with chemotherapy in comparison to chemotherapy alone. We also observed that treatment with azacitidine and panobinostat decreases the ability of ALL cells to effectively adhere to osteoblasts, suggesting a role that down-regulation of cell adhesion molecules (CAMs) may play in mediating this response. Due to the need for direct contact of ALL cells with osteoblasts for the chemoprotective effects, we investigated how treatment with azacitidine and panobinostat could affect the expression levels of certain CAMs. We found that N-cadherin was induced in ALL cells in coculture with osteoblasts and that increase was partially reversed by azacitidine and panobinostat. β-catenin, which is known to interact with N-cadherin, was also up-regulated in ALL cells cocultured with osteoblasts and was reversed by azacitidine and panobinostat. These data suggests that azacitidine and panobinostat may overcome microenvironment-induced chemoprotection by decreasing the expression of certain CAMs like N-cadherin and interfering with their downstream signaling pathways. Citation Format: Anthony Quagliano, Sonali Barwe, Anilkumar Gopalakrishnapillai. Epigenetic drug treatment overcomes osteoblast-induced chemoprotection by suppressing cell adhesion and related signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5870. doi:10.1158/1538-7445.AM2017-5870</jats:p

Modeling Transient Myeloproliferative Disorder Using Induced Pluripotent Stem Cells and CRISPR/Cas9 Technology

Down syndrome (DS) is recognized as one of the most important leukemia-predisposing syndromes. Specifically, 1-2% of DS children develop acute myeloid leukemia (AML) prior to age 5. AML in DS children (DS-AML) is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1 (GATA1s). Trisomy 21 and GATA1s together induce a transient myeloproliferative disorder (TMD) exhibiting pre-leukemic characteristics. Approximately thirty percent of these cases progress into DS-AML by acquisition of additional somatic mutations in a step-wise manner. We employed disease modeling in vitro by the use of customizable induced pluripotent stem cells (iPSCs) (7, 8) to generate a TMD model. Isogenic iPSC lines derived from the fibroblasts of a DS patient with trisomy 21 and with disomy 21 were used. We also obtained DS2-iPS10 (iPSCs derived from DS patient fibroblast) from Prof. George Daley, Children's Hospital, Harvard University (Boston, MA). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system with the indicated guide sequence (Fig. 1A) was used to introduce clinically relevant GATA1 mutation in both disomic and trisomic iPSC lines. A representative plot of TIDE (Tracking of Indels by Decomposition) analysis showing 98% allelic mutation frequency of a clone with 2 bp deletion at chromosomal level (Fig. 1B) correlated with sequence analysis using Basic Local Sequence Alignment Tool (BLAST) and Sanger sequencing chromatogram (Fig. 1C). This mutation resulted in the disruption of first initiation codon and thus prevented the production of full length GATA1 protein, while allowing the usage of second initiation codon at 84th position to generate GATA1s. GATA1 and GATA1s are not expressed in iPSCs. To determine the expression of GATA1s, we differentiated these mutant iPSC lines into hematopoietic stem cell progenitors (HSPCs) using hematopoietic differentiation kit (StemCell Technologies) following a protocol depicted in Fig. 1D. The HSPCs derived from two distinct clones of trisomic iPSCs showed expression of full-length GATA1 protein and GATA1 mutant HSPCs lacked the expression of full-length GATA1 as expected (Fig. 1E). These HSPCs expressed GATA1s. Given that trisomy 21 promotes hematopoietic differentiation, an increase in the percentage of erythroid, megakaryoid and myeloid population was observed in trisomy 21 HSPCs with full length GATA1 (Fig. 1F, compare bars 1 and 3 in each category). The expression of GATA1s reduced erythroid lineage cells whereas it augmented megakaryoid and myeloid lineages in both disomy 21 (compare red and blue bars 1 and 2) and trisomy 21 backgrounds (compare bars 3 and 4). HSPCs derived from trisomy 21 iPSCs with GATA1s exhibited more megakaryoid expansion compared to the GATA1s in disomy 21 background (Fig. 1F, compare bars 2 and 4), in agreement with the synergistic function of trisomy 21 and GATA1s in promoting TMD. Transplantation of HSPCs derived from GATA1 mutated trisomic iPSCS into NSG-SGM3 mice showed the presence of human CD45+ cells in peripheral blood at 12 weeks post cell injection (Fig. 1G). In conclusion, we have developed a model system representing TMD, which can be used for step-wise modeling of Down-syndrome AML by introducing additional mutations. Figure 1 Disclosures No relevant conflicts of interest to declare. </jats:sec

Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2·3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family $Zn^2+$-metalloprotease FtsH (M_sF_t_sH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti- E_cF_t_sH antibodies respectively. The recombinant and the native M_sF_t_sH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of M_sF_t_sH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The M_sf_t_sH gene did not complement lethality of a \delta ftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely \sigma 32 protein and the protein translocase subunit SecY, of E. coli cell