Biomedical subjects

A Rehemtulla

Publications and source records attributed to A Rehemtulla.

At least 37 records · Page 2Linked to original sources

PubMed1999

Toward an enzyme/prodrug strategy for cancer gene therapy: endogenous activation of carboxypeptidase A mutants by the PACE/Furin family of propeptidases.

In an effort to develop a gene-dependent enzyme/prodrug therapy (GDEPT) for tumor-specific delivery of methotrexate (MTX) we have chosen to construct mutant forms of carboxypeptidase A1 (CPA) that circumvent the requirement for trypsin-dependent activation. The basis of this strategy is that methotrexate-alpha-peptides are inefficient substrates for the reduced folate carrier (RFC) and hence cannot be internalized by cells. However, the blocking amino acid can be cleaved by CPA to liberate MTX, which is then internalized by the RFC, resulting in inhibition of dihydrofolate reductase and cytotoxicity. A battery of mutant CPAs was generated, in which the putative trypsin cleavage sites in the propeptide were mutated to the consensus recognition sequence for mammalian subtilisin-like propeptidases. These mutant forms of CPA were evaluated for expression, activation, and catalytic activity by transiently transfecting them into COS-1 cells both in the absence and in the presence of cotransfected propeptidases. CPA95 was identified as the most efficiently cleaved mutant, and further studies of this mutant indicated that the endogenously activated enzyme had kinetic parameters identical to those of the trypsin-activated wild-type protein. In addition, endogenously activated CPA95 could effectively sensitize cells to MTX-Phe in culture, decreasing the IC50 of MTX-Phe from 25- to 250-fold in squamous cell carcinoma cells expressing active CPA as compared with the parental lines.

Animals↗

PubMed1999

A caspase-resistant form of Bcl-X(L), but not wild type Bcl-X(L), promotes clonogenic survival after ionizing radiation.

Bcl-2 and Bcl-X(L) belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-X(L) overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-X(L) protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-X(L) to inhibit these markers of apoptosis. When Bcl-X(L) was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-X(L) was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-X(L) by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-X(L) (Bcl-X(L)-deltaloop). Cells overexpressing Bcl-X(L)-deltaloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-X(L)-deltaloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-X(L) overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-X(L) is not a prognostic marker of tumor response to cancer therapy.

Apoptosis↗

PubMed1999

Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation and for factor Xa activation.

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild-type, Q306-rFV, Q506-rFV, and Q306Q506-rFV. All had similar time courses for thrombin activation. Q506-rFVa was cleaved by APC at R306 and was moderately resistant to APC in plasma-clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma-derived Q506-FVa. Q306-rFVa was cleaved by APC at R506 and gave a low APC-resistance ratio similar to Q506-rFVa in clotting assays, whereas unactivated Q306-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306-rFVa was inactivated by only < or = 40% under conditions where Q506-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306-rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506-rFV was very resistant to cleavage and activation by FXa. Q306Q506-rFV appeared to bind FXa and inhibit FXa's ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa-binding sites upon cleavage at R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.

Animals↗

PubMed1999

Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix.

Fibrillin-1, the main component of 10-12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin.

Animals↗

PubMed1999

Combined radiation and enzyme/prodrug treatment for head and neck cancer in an orthotopic animal model.

In an effort to improve the therapeutic outcome for squamous cell cancer of the head and neck, we have used the enzyme cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC) as a means to deliver the chemotherapeutic agent 5-fluorouracil (5-FU) in a tumor-specific manner and have evaluated the use of this treatment in combination with external-beam radiation. Infection of SCCVII cells in culture with a CD-expressing retrovirus and treatment with 5-FC was cytotoxic depending on the time of treatment and dose of 5-FC. An orthotopic model of squamous cell cancer of the head and neck was used in vivo to study the CD/5-FC system both alone and with concurrent radiation due to the radiosensitizing properties that 5-FU generates in situ. Treated mice were imaged using magnetic resonance imaging (MRI), and their survival was evaluated. Neither 5-FU nor radiation either alone or combined provided a survival advantage. In contrast, 5-FC treatment prolonged survival and decreased tumor burden compared to control animals, but the tumors recurred after the treatment ceased. Finally, combined treatment with concurrent administration of 5-FC and radiation resulted in a synergistic decrease in tumor growth and enhanced survival over treatment with 5-FC or radiation alone.

Animals↗

PubMed1998

Expression of Bcl-XS alters cytokinetics and decreases clonogenic survival in K12 rat colon carcinoma cells.

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death, and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. To help understand these mechanisms we have established stable derivatives of the K12 rat colon carcinoma cell line that express bcl-XS in a tetracycline-regulated manner, using an autoregulatory retroviral cassette. When bcl-XS expression is induced, we observe two phenotypic responses. A small fraction of cells appear to undergo spontaneous apoptosis while the majority of cells undergo a form of cytostasis. In the latter case, the cells stop dividing (or divide a limited number of times at a retarded rate) and swell to many times their original size. These cells can take on a ghostlike appearance and subsequently detach from the culture plates and die or they may remain intact in a hindered state of proliferation. Doubling times were calculated to be 31.4 h in the presence of tetracycline and 50.4 h without tetracycline, bcl-XS expression also causes dramatic alterations in the cell cycle distribution of K12 cells manifesting as a substantial decrease (approximately 50%) in the fraction of S phase cells with a concomitant increase in the G1 population. Continuous expression of bcl-XS, at levels approximately equal to that of bcl-XL, decreased the viability of K12 cells as demonstrated by a log decline in clonogenic survival. This decrease occurred without considerable apoptosis or a compensatory increase in the level of bcl-XL. None of these phenotypes were present in control cells expressing beta-galactosidase in a similar retroviral cassette. These observations demonstrate that bcl-XS can have substantial cytokinetic effects under circumstances that produce relatively little apoptosis.

Animals↗

PubMed1998

Biochemical characterization and subcellular localization of the mouse retinitis pigmentosa GTPase regulator (mRpgr).

The retinitis pigmentosa GTPase regulator (RPGR) gene encodes a protein homologous to the RCC1 guanine nucleotide exchange factor and is mutated in 20% of patients with X-linked retinitis pigmentosa. We have characterized the full-length and variant cDNAs corresponding to the mouse homolog of the RPGR gene (mRpgr). Comparison with the human cDNA revealed sequence identity primarily in the region of RCC1 homology repeats. As in humans, the mRpgr gene maps within 50 kilobases from the 5'-end of the Otc gene. The mRpgr transcripts are detected as early as E7 during embryonic development and are expressed widely in the adult mice. Variant mRpgr isoforms are generated by alternative splicing and by utilizing two in-frame initiation codons. The products of mRpgr cDNAs migrate aberrantly in SDS-polyacrylamide gels because of a charged domain. In transfected COS cells, the mRpgr protein is isoprenylated and is localized in the Golgi complex. This subcellular distribution is not observed after treatments with brefeldin A or mevastatin and when the conserved isoprenylation sequence (CTIL) at the carboxyl terminus is deleted or mutagenized. These studies suggest a role for the mRpgr protein in Golgi transport and form the basis for investigating the mechanism of photoreceptor degeneration in X-linked retinitis pigmentosa.

Alternative Splicing↗

PubMed1998

Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine.

In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC. However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells. The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy. However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.

Animals↗

PubMed1998

Lack of cell surface Fas/APO-1 expression in pulmonary adenocarcinomas.

The Fas receptor and ligand initiate an apoptotic pathway. Alterations in this pathway within tumor cells can result in escape from apoptosis and immune surveillance. We evaluated Fas protein expression in 42 primary pulmonary adenocarcinomas, and Fas expression and function in the lung adenocarcinoma cell lines A549 and A427. Immunohistochemical analysis demonstrated Fas protein expression in 47.6% of the tumors; however, Fas-positive tumors demonstrated cytoplasmic staining without cell surface expression. Northern blot analysis indicated that levels of Fas mRNA were similar in Fas protein-positive tumors to levels in normal lung tissue, but were reduced in Fas protein-negative tumors. Soluble form Fas was not detected in the majority of these tumors either by RT-PCR or Western blot analysis. Cell surface Fas protein expression was minimal in A549 and A427 cell lines as determined by flow cytometry. Both cell lines demonstrated Fas mRNA expression by Northern blot analysis and abundant protein expression by Western blot analysis. Transfection of the Fas cDNA derived from A549 cells induced surface Fas protein in COS cells; however, stable transfection of a native Fas cDNA into A549 cells failed to induce surface Fas protein expression. Parental A549 cells and A549 cells transfected with a Fas expression vector were resistant to Fas-mediated apoptosis. Transgenic expression of a FLAG-tagged Fas cDNA in A549 cells, with visualization of the Fas-FLAG protein using confocal microscopy, demonstrated that the Fas-FLAG protein was retained within cytoplasmic portions of the cell and was not translocated to the cell surface. These findings suggest that the Fas protein is reduced or not present on the cell surface in the primary lung tumors and is sequestered within A549 tumorigenic lung cells, and these alterations directly affect the cells resistance to Fas-mediated apoptosis.

Adenocarcinoma↗

PubMed1997

Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma.

This study describes Fas (CD95) expression in Barrett's esophagus, adenocarcinomas of the esophagus, and three esophageal adenocarcinoma cell lines. Immunohistochemical analysis of Barrett's esophagus demonstrated cell surface expression of Fas protein. In contrast, 30.5% of esophageal adenocarcinomas examined by immunohistochemical analysis demonstrated faint cytoplasmic staining, and 69.5% were negative for Fas. Similar levels of Fas mRNA were identified in tumors compared to mRNA levels in esophageal squamous mucosa or Barrett's esophagus. An approximately Mr 48,000 Fas protein was identified by Western blot analysis in tumors that were negative for Fas expression by immunohistochemical analysis. The esophageal adenocarcinoma cell line Seg-1 was negative for Fas expression by immunohistochemical analysis, but Western blot analysis demonstrated abundant, appropriately sized Fas protein. In agreement with the immunohistochemical analysis, flow cytometry of Seg-1 showed minimal amounts of Fas on the cell surface, which correlated with resistance to Fas-mediated apoptosis. No mutations in the Seg-1 Fas coding sequence or exon 1 were identified by sequence analysis. This was confirmed by transient transfection of COS cells with expression vectors generated from the Seg-1 Fas cDNA, which resulted in cell surface expression of the Fas protein. Stable transfection of Seg-1 with a Fas expression vector did not result in efficient Fas expression on the cell surface. Seg-1 cells, transiently transfected with a Fas-FLAG expression vector and examined for protein expression using confocal microscopy and an anti-FLAG antibody, showed that the Fas-FLAG protein was not present on the cell surface but was present in the cytoplasm. Taken together, these results indicate that expression of Fas on the cell surface by esophageal adenocarcinoma is reduced. In an esophageal adenocarcinoma cell line, wild-type Fas protein is retained in the cytoplasm, and this correlates with resistance to Fas-mediated apoptosis. The retention of wild-type Fas protein within the cytoplasm may represent a mechanism by which malignant cells evade Fas-mediated apoptosis.

Adenocarcinoma↗

PubMed1997

Ultraviolet radiation-induced apoptosis is mediated by activation of CD-95 (Fas/APO-1).

Exposure to ultraviolet light (UV) can induce apoptosis in mammalian cells. The mechanism by which UV radiation engages the suicide apparatus is unclear. Here we demonstrate that UV radiation can activate the Fas pathway via receptor aggregation and subsequent recruitment of the death adaptor molecule FADD/MORT1. UV radiation-induced apoptosis was inhibited by both a dominant negative version of FADD (FADD-DN) and the caspase inhibitor CrmA. Thus, activation of the Fas pathway represents a physiologic mechanism by which UV-damaged cells are eliminated.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Protection of herpes simplex virus thymidine kinase-transduced cells from ganciclovir-mediated cytotoxicity by bystander cells: the Good Samaritan effect.

Although considerable attention has been directed in the field of gene therapy toward elucidating the mechanism by which a transduced cell could kill a bystander cell, little is known about how bystander cells may affect transduced cells. We hypothesized that bystander cells, particularly if they were capable of gap junctional communication, could protect cells transduced with the herpes simplex virus thymidine kinase (HSV-TK) from ganciclovir (GCV)-induced cytotoxicity. To test this hypothesis, we used a rat hepatocyte cell line (WB) that can carry out efficient gap junctional communication, a WB clone transduced with HSV-TK (WB-TK), and a communication-incompetent subclone of WB cells (aB1). We cocultured WB-TK cells with either WB or aB1 cells, treated them with GCV, and then plated the cells into selective media that permitted us to quantify independently the surviving fraction of WB-TK cells or bystander cells. We found that WB bystander cells conferred up to a 1000-fold protection on WB-TK cells treated with GCV. aB1 cells conferred detectable, but significantly less, protection. These findings demonstrate that herpes simplex virus thymidine kinase-transduced cells can be significantly protected by bystander cells, particularly those that can carry out gap junctional communication. Whether this "Good Samaritan" effect improves the overall efficacy of gene therapy, by prolonging the survival of the source of toxic metabolites, or decreases effectiveness by increasing the survival of transduced cells will need to be determined in vivo.

Animals↗

PubMed1997

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.

Animals↗

PubMed1996

The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures.

In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp. Activity of the -40 to -84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.

Amino Acid Sequence↗

PubMed1996

Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain.

Membrane type matrix metalloproteinase 1 (MT-MMP1), a novel 63-kDa member of the matrix metalloproteinase family, is a membrane-anchored enzyme and an activator for gelatinase A. In addition to its C-terminal hydrophobic transmembrane domain, MT-MMP1 has an insertion of 11 amino acids between its propeptide and catalytic domain encrypted with a RRKR recognition motif for the paired basic amino acid cleaving enzyme, furin. In this report, we investigated whether the cleavage of the RRKR motif of MT-MMP1 by Golgi-associated furin is analogous to a similar enzyme activation mechanism observed with stromelysin-3. Mutant forms of MT-MMP1 were cotransfected into COS-1 cells with cDNAs for pro-gelatinase A and/or furin. Immunoprecipitation and immunoblotting using specific antibodies were employed to characterize cell proteins. Whereas furin readily cleaved soluble MT-MMP1 lacking the transmembrane domain (DeltaMT-MMP1), a soluble stromelysin-1/DeltaMT-MMP1 chimera without the RRKR basic motif was resistant to furin-induced cleavage. COS-1 cells cotransfected with wild type MT-MMP1 cDNA and furin cDNA demonstrated a 63-kDa protein (latent enzyme) on SDS-polyacrylamide gel electrophoresis rather than the anticipated lower molecular weight activated enzyme. Inhibition of furin activity with alpha1-protease inhibitorPittsburgh (a furin inhibitor) did not affect the pro-gelatinase A activation mechanism in COS-1 cells cotransfected with MT-MMP1 and pro-gelatinase A cDNAs. Furthermore, substitution of the RRKR motif of MT-MMP1 with alanine residues by site-directed mutagenesis resulted in the same 63-kDa protein without loss of pro-gelatinase A activation function. These data indicate that furin-induced activation of MT-MMP1 is not a prerequisite for pro-gelatinase A activation. The mechanism of activation of cell-bound MT-MMP1 remains to be elucidated.

Animals↗

PubMed1996

A eukaryotic translation initiation factor 2-associated 67 kDa glycoprotein partially reverses protein synthesis inhibition by activated double-stranded RNA-dependent protein kinase in intact cells.

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.

Aminopeptidases↗

PubMed1996

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

Animals↗

PubMed1996

A common frameshift mutation in von Willebrand factor does not alter mRNA stability but interferes with normal propeptide processing.

Quantitative defects in von Willebrand factor (VWF) result in type 1 and type 3 von Willebrand disease (VWD). This study characterizes the defect in VWF expression resulting from a single nucleotide deletion in VWF exon 18, a mutation previously reported to be common among type 3 VWD patients. A severely affected (type 3) VWD patient in the current pedigree is homozygous for the mutation, whereas heterozygous individuals exhibit variable expression of type 1 VWD. In contrast to the previously reported high frequency of the exon 18 deletion in Sweden and Germany, this mutation appears to be infrequent among type 3 VWD patients in the United States. Although this frameshift mutation results in proximal premature termination of VWF translation, the abnormal VWF mRNA is stable. The mutant truncated recombinant VWF protein is retained within the transfected cell, and no propeptide processing is observed, suggesting a defect in protein folding. Cotransfection of mutant and wild-type recombinant VWF fails to demonstrate a dominant effect of the mutant on the normal allele. Consistent with these results, plasma VWF propeptide of the homozygous individual was markedly reduced whereas heterozygotes exhibited moderately reduced levels. In contrast to type 2A VWD (group 1), the misfolded mutant protein does not appear to exert a dominant-negative effect on normal VWF subunits expressed from the wild-type allele.

Exons↗