Assessment of normal and mutant human presenilin function in Caenorhabditis elegans

We provide evidence that normal human presenilins can substitute for Caenorhabditis elegans SEL-12 protein in functional assays in vivo. In addition, six familial Alzheimer disease-linked mutant human presenilins were tested and found to have reduced ability to rescue the sel-12 mutant phenotype, suggesting that they have lower than normal presenilin activity. A human presenilin 1 deletion variant that fails to be proteolytically processed and a mutant SEL-12 protein that lacks the C terminus display considerable activity in this assay, suggesting that neither presenilin proteolysis nor the C terminus is absolutely required for normal presenilin function. We also show that sel-12 is expressed in most neural and nonneural cell types in all developmental stages. The reduced activity of mutant presenilins and as yet unknown gain-of-function properties may be a contributing factor in the development of Alzheimer disease.

Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Human sulfite oxidase R160Q: Identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of β-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gusmps/mps) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human β-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gusmps/mps) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gusmps/mps) mouse. However, they are now tolerant to immune challenge with human β-glucuronidase. This “tolerant MPS VII mouse model” should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.

Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues.

The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986]. Systematic replacement of hydrophobic residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.

An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele.

If deprived of wild-type p53 function, the body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.