Biomedical scientist with an interest in the mechanisms regulating lipid metabolism, especially how metabolic disturbances affect the development of human disease
Homogenates and microsomal fractions prepared from biopsies of highly differentiated human hepato... more Homogenates and microsomal fractions prepared from biopsies of highly differentiated human hepatocellular carcinomas were found to contain low levels of dolichol in comparison with control tissue. In contrast, the amount of dolichyl phosphate in tumor homogenates was unchanged and actually increased in the microsomal fraction. The pattern of individual polyisoprenoids, both in the free and the phosphorylated dolichol fractions of hepatomas, did not exhibit any major alterations compared to the control. The rates of incorporation of [3H]mevalonic acid into dolichol and dolichyl phosphate in hepatomas were low. The dolichol monophosphatase activities in microsomal fractions from hepatomas and controls did not show any major differences, whereas the activity of the CTP-dependent dolichol kinase was increased in tumor microsomes. Glycosylation of endogenous dolichyl phosphate and of total protein using certain nucleotide-activated sugars was found to be slightly elevated in microsomal f...
Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized... more Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient’s mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense muta...
The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates c... more The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates cholesterol, fatty acid, and triglyceride synthesis and metabolism. However, they are also targeted by the ubiquitin ligase Fbw7, a major tumor suppressor, suggesting that they could regulate cell growth. Indeed, enhanced lipid synthesis is a hallmark of many human tumors. Thus, the SREBP pathway has recently emerged as a potential target for cancer therapy. We have previously demonstrated that one of these transcription factors, SREBP1, is stabilized and remains associated with target promoters during mitosis, suggesting that the expression of these target genes could be important as cells enter G1 and transcription is restored. Activation of cyclin D-cdk4/6 complexes is critical for the phosphorylation and inactivation of the retinoblastoma protein (Rb) family of transcriptional repressors and progression through the G1 phase of the cell cycle. Importantly, the cyclin D-cdk4/6-Rb regulatory axis is frequently dysregulated in human cancer. In the current manuscript, we demonstrate that SREBP1 activates the expression of cyclin D1, a coactivator of cdk4 and cdk6, by binding to an E-box in the cyclin D1 promoter. Consequently, inactivation of SREBP1 in human liver and breast cancer cell lines reduces the expression of cyclin D1 and attenuates Rb phosphorylation. Rb phosphorylation in these cells can be rescued by restoring cyclin D1 expression. On the other hand, expression of active SREBP1 induced the expression of cyclin D1 and increased the phosphorylation of Rb in a manner dependent on cyclin D1 and cdk4/6 activity. Inactivation of SREBP1 resulted in reduced expression of cyclin D1, attenuated phosphorylation of Rb, and reduced proliferation. Inactivation of SREBP1 also reduced the insulin-dependent regulation of the cyclin D1 gene. At the same time, SREBP1 is known to play an important role in supporting lipid synthesis in cancer cells. Thus, we propose that the SREBP1-dependent regulation of cyclin D1 coordinates cell proliferation with the enhanced lipid synthesis required to support cell growth.
The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synth... more The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.
The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cho... more The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cholesterol and fatty acid synthesis and metabolism. There are three SREBP proteins, SREBP1a, SREBP1c and SREBP2, with SREBP1a being the strongest transcription factor. The expression of SREBP1a is restricted to rapidly proliferating cells, including cancer cells. The SREBP proteins are translated as large, inactive precursors bound to the endoplasmic reticulum (ER) membranes. These precursors undergo a two-step cleavage process that releases the amino terminal domains of the proteins, which translocate to the nucleus and function as transcription factors. The nuclear forms of the SREBPs are rapidly degraded by the ubiquitin-proteasome system in a manner dependent on the Fbw7 ubiquitin ligase. Consequently, inactivation of Fbw7 results in the stabilization of active SREBP1 and SREBP2 and enhanced expression of target genes. We report that the inactivation of Fbw7 in cancer cells blocks the ...
Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized... more Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized by the accumulation of glycogen mainly in the liver. It is inherited in an autosomal recessive manner due to mutations in the SLC2A2 gene. SLC2A2 encodes for the glucose transporter GLUT2 and is expressed in tissues that are involved in glucose homeostasis. The molecular mechanisms of dysglycemia in FBS are still not clearly understood. In this study, we report two cases of FBS with classical phenotypes of FBS associated with dysglycemia. Genomic DNA was extracted and analyzed by whole-genome and Sanger sequencing, and patient PBMCs were used for molecular analysis. One patient had an exonic SLC2A2 mutation (c.1093C > T in exon 9, R365X), while the other patient had a novel intronic SLC2A2 mutation (c.613-7T>G). Surprisingly, the exonic mutation resulted in the overexpression of dysfunctional GLUT2, resulting in the dysregulated expression of other glucose transporters. The intron...
Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on tw... more Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on two cis elements in the proximal promoter. These elements, an inverted CCAAT box and sterol regulatory element 3 (SRE-3), bind NF-Y and sterol regulatory element-binding protein 1 (SREBP-1), respectively. We now demonstrate that the binding of recombinant SREBP-1 to its cognate site (SRE-3) within the FPP synthase promoter in vitro is enhanced by binding of NF-Y to the upstream inverted CCAAT box. Using an FPP synthase promoter fragment containing the binding sites for both NF-Y and SREBP-1 in gel mobility shift assays, we demonstrate that the addition of NF-Y increases the binding of SREBP-1 to SRE-3 over 20-fold. In contrast, NF-Y does not stimulate the binding of SREBP-1 to SRE-3 when the inverted CCAAT box is either mutated or 4 base pairs (bp) are inserted between the inverted CCAAT box and SRE-3. Promoter-reporter genes, containing either the wildtype FPP synthase promoter sequence or containing the 4-bp insertion between the inverted CCAAT box and SRE-3, were transiently transfected into cells. The activity of the wild-type promoter-reporter gene increased when the cells were either incubated in sterol-depleted medium or were co-transfected with an expression vector encoding transcriptionally active SREBP-1. This increase in activity was attenuated when the promoter contained the 4-bp insert, consistent with defective binding of SREBP to the promoter in vivo. These studies suggest that the binding of SREBP-1 to SRE-3 in the FPP synthase promoter, and subsequent stimulation of transcription, is dependent on synergistic binding and a functional interaction between SREBP-1 and NF-Y.
Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diph... more Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.
We demonstrate that the mRNA levels of glycerol-3phosphate acyltransferase (GPAT), a mitochondria... more We demonstrate that the mRNA levels of glycerol-3phosphate acyltransferase (GPAT), a mitochondrial enzyme catalyzing the initial step in glycerolipid synthesis, are induced during the differentiation of 3T3-L1 preadipocytes to adipocytes and following ectopic expression of rat adipocyte determination and differentiation factor 1 (ADD1), a protein with high homology to the human sterol regulatory element-binding protein-1 (SREBP-1). The increase in GPAT mRNA levels that occurs during differentiation is partially prevented by ectopic expression of a dominant negative form of ADD1. Nucleotide sequences corresponding to the proximal promoter of the murine mitochondrial GPAT gene (Jerkins, A.
Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M a... more Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M and >70% of the aphidicolin-arrested cells were in S phase (not depicted). (B) The indicated Hct116 cell clones were arrested in S phase with a sequential thymidine/hydroxyurea block and released for the indicated time periods. The abundance of cyclin E, phosphorylated cyclin E species, and Grb2 (loading control) are shown. (C) Hct116 cells were treated as in A and lysates were examined for Fbw7 and cyclin E expression. Fbw7-null cell lysates serve as negative control. (D) Hct116 cells were arrested and released from aphidicolin and Fbw7 abundance was determined.<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
(B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were imm... more (B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were immunoblotted for c-Myc. (C) Real-time PCR analysis of c-Myc mRNA in the indicated Hct116 cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates). (D) Lysates from the indicated Hct116 cells were immunoprecipitated with anti-SREBP1 antibody and immunoblotted for either total SREBP1 (left) or pT426 SREBP1 (right).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
(B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg e... more (B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg each of Fbw7β and Fbw7γ) and treated with cycloheximide (CHX) as indicated. The three isoforms are indicated. (C) Schematic demonstrating locations of inserted stop codons for each knockout genotype (see text). (D) Lysates were prepared from confluent p150 plates of Hct116 clones of the indicated genotypes and immunoprecipitated with polyclonal anti-Fbw7 antibody followed by immunoblotting with monoclonal anti-Fbw7 antibody. (E) Real-time PCR analysis of relative Fbw7 isoform mRNA expression in Hct116 Fbw7 knockout cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isopre... more Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with K,,, values of 24 and 5 pM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent K , of 36 FM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyll2-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.
The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation... more The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation (c.3999 G > A) was not mediated through autoimmunity. We have evaluated the ability of the proband’s T cells to be activated by assessing their CTLA-4 expression. A nonsignificant difference was seen in the CTLA-4 expression on CD3+ T cells compared to the healthy control at basal level and after stimulation with PMA/ionomycin. Blood transcriptomic analysis have shown a remarkable increase in abundance of transcripts related to CD71+ erythroid cells. There were no differences in the expression of modules related to autoimmunity diseases between the proband and pooled healthy controls. In addition, our novel findings show that siRNA knockdown of LRBA in mouse pancreatic β-cells leads reduced cellular proinsulin, insulin and consequently insulin secretion, without change in cell viability in cultured MIN6 cells.
Ere treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicate... more Ere treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicated times. Cyclin E abundance and activity, as well as cyclin A abundance (a marker of cell cycle synchronization) are shown (*, background band). (B) Hct116 cells or Fbw7-null Hct116 cells were transfected with either control or p21Cip1-specific siRNAs and, after 48 h, treated with aphidicolin (Aph) or nocodazole (Noc). Cyclin E abundance, activity, and S384 phosphorylation are shown. The efficacy of p21 knockdown is shown. (C) Fbw7-null Hct116 cells were treated with aphidicolin or nocodazole and lysates were immunoprecipitated with anti–cyclin E antibody. The amount of CDK2 and Y15–phosphorylated CDK2 bound to cyclin E is shown. (D) Increased Fbw7 binding of cyclin E obtained from mitotic Fbw7-null lysates. Compare the amount of cyclin E that coprecipitates with Fbw7 in lanes 4 and 5 (middle).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent sub...
Background: The objective of this study was to develop a protocol for direct use of saliva in tes... more Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers...
Box 595 blocking the interactions between the R-Smads and the activated receptors (Hayashi et al.... more Box 595 blocking the interactions between the R-Smads and the activated receptors (Hayashi et al., 1997; Nakao et al., Husargatan 3 S-751 24 Uppsala 1997). Second, Smad7 interacts with the E3-ubiquitin ligases Smurf1 or Smurf2 in the nucleus; after TGF Sweden stimulation, the Smad7-Smurf complex translocates from the nucleus to the plasma membrane, where Smurf induces ubiquitination and degradation of the TGF re-Summary ceptors (Ebisawa et al., 2001; Kavsak et al., 2000). Acetylation is a dynamic posttranslational modifica-Smad proteins regulate gene expression in response to TGF signaling. Here we present evidence that tion of lysine residues. Proteins with intrinsic histone acetyltransferase (HAT) activity act as transcriptional Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine coactivators by acetylating histones and thereby induce an open chromatin conformation, which allows the tran-residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects scriptional machinery access to promoters (Roth et al., 2001). The best characterized HATs are p300, CBP, and it from TGF-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 P/CAF (Roth et al., 2001). These proteins interact with a large number of transcription factors, such as the also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquiti
Homogenates and microsomal fractions prepared from biopsies of highly differentiated human hepato... more Homogenates and microsomal fractions prepared from biopsies of highly differentiated human hepatocellular carcinomas were found to contain low levels of dolichol in comparison with control tissue. In contrast, the amount of dolichyl phosphate in tumor homogenates was unchanged and actually increased in the microsomal fraction. The pattern of individual polyisoprenoids, both in the free and the phosphorylated dolichol fractions of hepatomas, did not exhibit any major alterations compared to the control. The rates of incorporation of [3H]mevalonic acid into dolichol and dolichyl phosphate in hepatomas were low. The dolichol monophosphatase activities in microsomal fractions from hepatomas and controls did not show any major differences, whereas the activity of the CTP-dependent dolichol kinase was increased in tumor microsomes. Glycosylation of endogenous dolichyl phosphate and of total protein using certain nucleotide-activated sugars was found to be slightly elevated in microsomal f...
Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized... more Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient’s mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense muta...
The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates c... more The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates cholesterol, fatty acid, and triglyceride synthesis and metabolism. However, they are also targeted by the ubiquitin ligase Fbw7, a major tumor suppressor, suggesting that they could regulate cell growth. Indeed, enhanced lipid synthesis is a hallmark of many human tumors. Thus, the SREBP pathway has recently emerged as a potential target for cancer therapy. We have previously demonstrated that one of these transcription factors, SREBP1, is stabilized and remains associated with target promoters during mitosis, suggesting that the expression of these target genes could be important as cells enter G1 and transcription is restored. Activation of cyclin D-cdk4/6 complexes is critical for the phosphorylation and inactivation of the retinoblastoma protein (Rb) family of transcriptional repressors and progression through the G1 phase of the cell cycle. Importantly, the cyclin D-cdk4/6-Rb regulatory axis is frequently dysregulated in human cancer. In the current manuscript, we demonstrate that SREBP1 activates the expression of cyclin D1, a coactivator of cdk4 and cdk6, by binding to an E-box in the cyclin D1 promoter. Consequently, inactivation of SREBP1 in human liver and breast cancer cell lines reduces the expression of cyclin D1 and attenuates Rb phosphorylation. Rb phosphorylation in these cells can be rescued by restoring cyclin D1 expression. On the other hand, expression of active SREBP1 induced the expression of cyclin D1 and increased the phosphorylation of Rb in a manner dependent on cyclin D1 and cdk4/6 activity. Inactivation of SREBP1 resulted in reduced expression of cyclin D1, attenuated phosphorylation of Rb, and reduced proliferation. Inactivation of SREBP1 also reduced the insulin-dependent regulation of the cyclin D1 gene. At the same time, SREBP1 is known to play an important role in supporting lipid synthesis in cancer cells. Thus, we propose that the SREBP1-dependent regulation of cyclin D1 coordinates cell proliferation with the enhanced lipid synthesis required to support cell growth.
The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synth... more The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.
The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cho... more The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cholesterol and fatty acid synthesis and metabolism. There are three SREBP proteins, SREBP1a, SREBP1c and SREBP2, with SREBP1a being the strongest transcription factor. The expression of SREBP1a is restricted to rapidly proliferating cells, including cancer cells. The SREBP proteins are translated as large, inactive precursors bound to the endoplasmic reticulum (ER) membranes. These precursors undergo a two-step cleavage process that releases the amino terminal domains of the proteins, which translocate to the nucleus and function as transcription factors. The nuclear forms of the SREBPs are rapidly degraded by the ubiquitin-proteasome system in a manner dependent on the Fbw7 ubiquitin ligase. Consequently, inactivation of Fbw7 results in the stabilization of active SREBP1 and SREBP2 and enhanced expression of target genes. We report that the inactivation of Fbw7 in cancer cells blocks the ...
Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized... more Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized by the accumulation of glycogen mainly in the liver. It is inherited in an autosomal recessive manner due to mutations in the SLC2A2 gene. SLC2A2 encodes for the glucose transporter GLUT2 and is expressed in tissues that are involved in glucose homeostasis. The molecular mechanisms of dysglycemia in FBS are still not clearly understood. In this study, we report two cases of FBS with classical phenotypes of FBS associated with dysglycemia. Genomic DNA was extracted and analyzed by whole-genome and Sanger sequencing, and patient PBMCs were used for molecular analysis. One patient had an exonic SLC2A2 mutation (c.1093C > T in exon 9, R365X), while the other patient had a novel intronic SLC2A2 mutation (c.613-7T>G). Surprisingly, the exonic mutation resulted in the overexpression of dysfunctional GLUT2, resulting in the dysregulated expression of other glucose transporters. The intron...
Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on tw... more Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on two cis elements in the proximal promoter. These elements, an inverted CCAAT box and sterol regulatory element 3 (SRE-3), bind NF-Y and sterol regulatory element-binding protein 1 (SREBP-1), respectively. We now demonstrate that the binding of recombinant SREBP-1 to its cognate site (SRE-3) within the FPP synthase promoter in vitro is enhanced by binding of NF-Y to the upstream inverted CCAAT box. Using an FPP synthase promoter fragment containing the binding sites for both NF-Y and SREBP-1 in gel mobility shift assays, we demonstrate that the addition of NF-Y increases the binding of SREBP-1 to SRE-3 over 20-fold. In contrast, NF-Y does not stimulate the binding of SREBP-1 to SRE-3 when the inverted CCAAT box is either mutated or 4 base pairs (bp) are inserted between the inverted CCAAT box and SRE-3. Promoter-reporter genes, containing either the wildtype FPP synthase promoter sequence or containing the 4-bp insertion between the inverted CCAAT box and SRE-3, were transiently transfected into cells. The activity of the wild-type promoter-reporter gene increased when the cells were either incubated in sterol-depleted medium or were co-transfected with an expression vector encoding transcriptionally active SREBP-1. This increase in activity was attenuated when the promoter contained the 4-bp insert, consistent with defective binding of SREBP to the promoter in vivo. These studies suggest that the binding of SREBP-1 to SRE-3 in the FPP synthase promoter, and subsequent stimulation of transcription, is dependent on synergistic binding and a functional interaction between SREBP-1 and NF-Y.
Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diph... more Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.
We demonstrate that the mRNA levels of glycerol-3phosphate acyltransferase (GPAT), a mitochondria... more We demonstrate that the mRNA levels of glycerol-3phosphate acyltransferase (GPAT), a mitochondrial enzyme catalyzing the initial step in glycerolipid synthesis, are induced during the differentiation of 3T3-L1 preadipocytes to adipocytes and following ectopic expression of rat adipocyte determination and differentiation factor 1 (ADD1), a protein with high homology to the human sterol regulatory element-binding protein-1 (SREBP-1). The increase in GPAT mRNA levels that occurs during differentiation is partially prevented by ectopic expression of a dominant negative form of ADD1. Nucleotide sequences corresponding to the proximal promoter of the murine mitochondrial GPAT gene (Jerkins, A.
Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M a... more Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M and >70% of the aphidicolin-arrested cells were in S phase (not depicted). (B) The indicated Hct116 cell clones were arrested in S phase with a sequential thymidine/hydroxyurea block and released for the indicated time periods. The abundance of cyclin E, phosphorylated cyclin E species, and Grb2 (loading control) are shown. (C) Hct116 cells were treated as in A and lysates were examined for Fbw7 and cyclin E expression. Fbw7-null cell lysates serve as negative control. (D) Hct116 cells were arrested and released from aphidicolin and Fbw7 abundance was determined.<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
(B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were imm... more (B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were immunoblotted for c-Myc. (C) Real-time PCR analysis of c-Myc mRNA in the indicated Hct116 cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates). (D) Lysates from the indicated Hct116 cells were immunoprecipitated with anti-SREBP1 antibody and immunoblotted for either total SREBP1 (left) or pT426 SREBP1 (right).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
(B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg e... more (B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg each of Fbw7β and Fbw7γ) and treated with cycloheximide (CHX) as indicated. The three isoforms are indicated. (C) Schematic demonstrating locations of inserted stop codons for each knockout genotype (see text). (D) Lysates were prepared from confluent p150 plates of Hct116 clones of the indicated genotypes and immunoprecipitated with polyclonal anti-Fbw7 antibody followed by immunoblotting with monoclonal anti-Fbw7 antibody. (E) Real-time PCR analysis of relative Fbw7 isoform mRNA expression in Hct116 Fbw7 knockout cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"The Journal of Cell Biology 2008;181(6):913-920.Published online 16 Jun 2008PMCID:PMC2426948.
Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isopre... more Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with K,,, values of 24 and 5 pM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent K , of 36 FM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyll2-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.
The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation... more The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation (c.3999 G > A) was not mediated through autoimmunity. We have evaluated the ability of the proband’s T cells to be activated by assessing their CTLA-4 expression. A nonsignificant difference was seen in the CTLA-4 expression on CD3+ T cells compared to the healthy control at basal level and after stimulation with PMA/ionomycin. Blood transcriptomic analysis have shown a remarkable increase in abundance of transcripts related to CD71+ erythroid cells. There were no differences in the expression of modules related to autoimmunity diseases between the proband and pooled healthy controls. In addition, our novel findings show that siRNA knockdown of LRBA in mouse pancreatic β-cells leads reduced cellular proinsulin, insulin and consequently insulin secretion, without change in cell viability in cultured MIN6 cells.
Ere treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicate... more Ere treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicated times. Cyclin E abundance and activity, as well as cyclin A abundance (a marker of cell cycle synchronization) are shown (*, background band). (B) Hct116 cells or Fbw7-null Hct116 cells were transfected with either control or p21Cip1-specific siRNAs and, after 48 h, treated with aphidicolin (Aph) or nocodazole (Noc). Cyclin E abundance, activity, and S384 phosphorylation are shown. The efficacy of p21 knockdown is shown. (C) Fbw7-null Hct116 cells were treated with aphidicolin or nocodazole and lysates were immunoprecipitated with anti–cyclin E antibody. The amount of CDK2 and Y15–phosphorylated CDK2 bound to cyclin E is shown. (D) Increased Fbw7 binding of cyclin E obtained from mitotic Fbw7-null lysates. Compare the amount of cyclin E that coprecipitates with Fbw7 in lanes 4 and 5 (middle).<b>Copyright information:</b>Taken from "Isoform- and cell cycle–dependent sub...
Background: The objective of this study was to develop a protocol for direct use of saliva in tes... more Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers...
Box 595 blocking the interactions between the R-Smads and the activated receptors (Hayashi et al.... more Box 595 blocking the interactions between the R-Smads and the activated receptors (Hayashi et al., 1997; Nakao et al., Husargatan 3 S-751 24 Uppsala 1997). Second, Smad7 interacts with the E3-ubiquitin ligases Smurf1 or Smurf2 in the nucleus; after TGF Sweden stimulation, the Smad7-Smurf complex translocates from the nucleus to the plasma membrane, where Smurf induces ubiquitination and degradation of the TGF re-Summary ceptors (Ebisawa et al., 2001; Kavsak et al., 2000). Acetylation is a dynamic posttranslational modifica-Smad proteins regulate gene expression in response to TGF signaling. Here we present evidence that tion of lysine residues. Proteins with intrinsic histone acetyltransferase (HAT) activity act as transcriptional Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine coactivators by acetylating histones and thereby induce an open chromatin conformation, which allows the tran-residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects scriptional machinery access to promoters (Roth et al., 2001). The best characterized HATs are p300, CBP, and it from TGF-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 P/CAF (Roth et al., 2001). These proteins interact with a large number of transcription factors, such as the also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquiti
Uploads
Papers by Johan Ericsson