Nonidet P-40 Research Articles

Optimization of RNA Isolation from Brittle Leaf Disease Affected Date Palm Leaves and Construction of a Subtractive cDNA Library

A simple and efficient method was described here for the isolation of high-quality RNA from date palm leaves affected with Brittle Leaf Disease (BLD) and containing high amount of phenolic compounds. The procedure was based on the use of a non-ionic detergent Nonidet-P40 (NP-40), Polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in the extraction buffer in order to isolate cytoplasmic RNA and to prevent the oxidation of phenolic compounds. This method allowed the isolation of intact RNA, suitable for cDNA synthesis and library construction. Differential screening of the subtractive cDNA library from affected leaf RNA led to the identification of some BLD-induced genes.

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea-urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.

The HIV-1 p6/EIAV p9 docking site in Alix is autoinhibited as revealed by a conformation-sensitive anti-Alix monoclonal antibody

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6(Gag) or EIAV (equine infectious anaemia virus) p9(Gag), and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6(Gag)/p9(Gag) and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active p6(Gag)/p9(Gag) docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6(Gag)/p9(Gag) docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.

Evaluation of an Effective Sample Prefractionation Method for the Proteome Analysis of Breast Cancer Tissue Using Narrow Range Two-Dimensional Gel Electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.

Phosphorylation Regulates Tau Interactions with Src Homology 3 Domains of Phosphatidylinositol 3-Kinase, Phospholipase Cγ1, Grb2, and Src Family Kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.

Distinct Effects of N-Acetylgalactosamine-4-sulfatase and Galactose-6-sulfatase Expression on Chondroitin Sulfates

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

Biochemical characterization of two -mannosidases from breadfruit (Artocarpus communis) seeds

The importance of α -mannosidases in glycoproteins processing and their application in food and pharmaceutical industry led us to further explore plant α -mannosidases. Thus, two α -mannosidases were purified from matured breadfruit (Artocarpus communis) seeds, by successive chromatography on Diethylaminoethyl-Sepharose CL-6B and Sephacryl S-200 HR to apparent homogeneity. The two isoenzymes named Ma and Mb had native molecular weights of approximately 75 and 60 kDa, respectively. Sodium Dodecyl Sulfate- polyacrylamide gel electrophoresis of these α -mannosidases resolved a single protein band with molecular weights estimated to be 75 kDa for isoform Ma and 61 kDa for Mb. Breadfruitα -mannosidases had optima pH (5.6) and temperature (60°C), and appeared to be stable in presence of some detergents such as Hexansulfonic acid sodium salt, Polyoxyethylen-9-lauryl ether, Nonidet P40, Triton X-100 as well as Ca2+ and Zn2+. The effect of α -mannosidase inhibitors on the two isoenzymes showed that swainsonine and 1,4-dideoxy-1,4-imino mannitol at 0.01 mM totally inhibited their hydrolytic activity, while kifunensine and deoxymannojirimycin at the same concentration had no effect on these enzymes. Substrate specificity tests revealed that the enzymes exerted only α -mannosidase activity and cleaved α -(1,2); α -(1,3) and α -(1,6) linked mannobiose. Since breadfruit seed α -mannosidases were sensitive to furanose transition state analogs such as swainsonine and 1,4-dideoxy-1,4-imino mannitol and showed broad substrate specificity, these enzymes would belong to class II α -mannosidases. Key words: α -mannosidases, Artocarpus communis, breadfruit, purification, characterization.

A Protein Kinase Cε-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

Transforming Growth Factor-β Receptor Type I-dependent Fibrogenic Gene Program Is Mediated via Activation of Smad1 and ERK1/2 Pathways

The transforming growth factor (TGF)-beta/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-beta signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-betaRI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-betaRI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-betaRI, (ii) a TGF-betaRI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-betaRI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-betaRI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGFbeta-RI. Additional experiments demonstrated that a GC-rich response element located -6 to -16 (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc fibroblasts. In conclusion, this study defines a novel ALK1/Smad1- and ERK1/2-dependent, Smad3-independent mode of TGF-beta signaling that may operate during chronic stages of fibrosis in SSc.

Two Yeast PUF Proteins Negatively Regulate a Single mRNA

mRNA stability and translation are regulated by protein repressors that bind 3'-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3'-untranslated region-mediated control.

PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p

PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Prion Protein Helix1 Promotes Aggregation but Is Not Converted into β-Sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).

Defining the Requirements for Hsp40 and Hsp70 in the Hsp90 Chaperone Pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.

Hypothyroidism Reduces Tricarboxylate Carrier Activity and Expression in Rat Liver Mitochondria by Reducing Nuclear Transcription Rate and Splicing Efficiency

The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by approximately 40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3'-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels.

Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options

How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.