Gain Of Function Mechanism Research Articles

Autosomal dominant neuronal ceroid lipofuscinosis: Clinical features and molecular basis.

The neuronal ceroid lipofuscinoses (NCLs) are at least 13 distinct progressive neurodegenerative disorders unified by the accumulation of lysosomal auto-fluorescent material called lipofuscin. The only form that occurs via autosomal-dominant inheritance exhibits adult onset and is sometimes referred to as Parry type NCL. The manifestations may include behavioral symptoms followed by seizures, ataxia, dementia, and early death. Mutations in the gene DNAJC5 that codes for the presynaptic co-chaperone cysteine string protein-α (CSPα) were recently reported in sporadic adult-onset cases and in families with dominant inheritance. The mutant CSPα protein may lead to disease progression by both loss and gain of function mechanisms. Iron chelation therapy may be considered as a possible pharmaceutical intervention based on our recent mechanism-based proposal of CSPα oligomerization via ectopic Fe-S cluster-binding, summarized in this review.

De novo missense variants in the RAP1B gene identified in two patients with syndromic thrombocytopenia.

We present two independent cases of syndromic thrombocytopenia with multiple malformations, microcephaly, learning difficulties, dysmorphism and other features. Exome sequencing identified two novel de novo heterozygous variants in these patients, c.35G>T p.(Gly12Val) and c.178G>C p.(Gly60Arg), in the RAP1B gene (NM_001010942.2). These variants have not been described previously as germline variants, however functional studies in literature strongly suggest a clinical implication of these two activating hot spot positions. We hypothesize that pathogenic missense variants in the RAP1B gene cause congenital syndromic thrombocytopenia with a spectrum of associated malformations and dysmorphism, possibly through a gain of function mechanism.

Targeting codon 158 p53-mutant cancers via the induction of p53 acetylation

Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.

Recapitulation of the EEF1A2 D252H neurodevelopmental disorder-causing missense mutation in mice reveals a toxic gain of function.

Heterozygous de novo mutations in EEF1A2, encoding the tissue-specific translation elongation factor eEF1A2, have been shown to cause neurodevelopmental disorders including often severe epilepsy and intellectual disability. The mutational profile is unusual; ~50 different missense mutations have been identified but no obvious loss of function mutations, though large heterozygous deletions are known to be compatible with life. A key question is whether the heterozygous missense mutations operate through haploinsufficiency or a gain of function mechanism, an important prerequisite for design of therapeutic strategies. In order both to address this question and to provide a novel model for neurodevelopmental disorders resulting from mutations in EEF1A2, we created a new mouse model of the D252H mutation. This mutation causes the eEF1A2 protein to be expressed at lower levels in brain but higher in muscle in the mice. We compared both heterozygous and homozygous D252H and null mutant mice using behavioural and motor phenotyping alongside molecular modelling and analysis of binding partners. Although the proteomic analysis pointed to a loss of function for the D252H mutant protein, the D252H homozygous mice were more severely affected than null homozygotes on the same genetic background. Mice that are heterozygous for the missense mutation show no behavioural abnormalities but do have sex-specific deficits in body mass and motor function. The phenotyping of our novel mouse lines, together with analysis of molecular modelling and interacting proteins, suggest that the D252H mutation results in a gain of function.

How mutations shape p53 interactions with the genome to promote tumorigenesis and drug resistance.

The tumor suppressive transcription factor p53 regulates a wide array of cellular processes that confer upon cells an essential protection against cancer development. Wild-type p53 regulates gene expression by directly binding to DNA in a sequence-specific manner. p53 missense mutations are the most common mutations in malignant cells and can be regarded as synonymous with anticancer drug resistance and poor prognosis. The current review provides an overview of how the extraordinary variety of more than 2000 different mutant p53 proteins, known as the p53 mutome, affect the interaction of p53 with DNA. We discuss how the classification of p53 mutations to loss of function (LOF), gain of function (GOF), and dominant-negative (DN) inhibition of a remaining wild-type allele, hides a complex p53 mutation spectrum that depends on the distinctive nature of each mutant protein, requiring different therapeutic strategies for each mutant p53 protein. We propose to regard the different mutant p53 categories as continuous variables, that may not be independent of each other. In particular, we suggest here to consider GOF mutations as a special subset of LOF mutations, especially when mutant p53 binds to DNA through cooperation with other transcription factors, and we present a model for GOF mechanism that consolidates many observations on the GOF phenomenon. We review how novel mutant p53 targeting approaches aim to restore a wild-type-like DNA interaction and to overcome resistance to cancer therapy.

The Role of Mediator Complex Subunit 12 in Leiomyoma Biology

Uterine Leiomyomas are one of the most common clinically relevant solid tumors occurring in women of reproductive age. Until recently not much was known about the genetic etiology of these tumors. This review focusses on some of the past and present findings which have greatly contributed to a better understanding of the genetics of uterine leiomyomas. Next generation sequencing has shown that MED12 exon 2 variants are recurrently mutated in about 70 % of human uterine leiomyomas, implying a role of MED12 in leiomyoma biology. We further discuss the development of Med12 exon 2 variant models validating a role of these variants in leiomyoma causation via gain of function mechanism. Evidence from genomic studies and animal models strongly supports a role of MED12 in leiomyoma causation. The Med12 mouse models not only provide mechanistic insights but also provide a valuable platform to develop new treatment strategies against leiomyomas as an alternative to hysterectomy.

Gain-of-function miRNA signature by mutant p53 associates with poor cancer outcome.

Missense mutation of p53 not only impairs its tumor suppression function, but also causes oncogenic gain of function (GOF). The molecular underpinning of mutant p53 (mutp53) GOF is not fully understood, especially for the potential roles of non-coding genes. Here we identify the microRNA expression profile (microRNAome) of mutp53 on Arg282 by controlled microarray experiments, and clarify the prognostic significance of mutp53-regulated miRNAs in cancers. A predominant repression effect on miRNA expression was found for mutant p53, with 183 significantly downregulated and only 12 upregulated miRNAs. Mutp53 and wild-type (wtp53) commonly upregulate let-7i, and other two miRNAs were upregulated by wtp53 but repressed by mutp53 (miR-610 and miR-3065–3p). Based the mutp53-regulated miRNA signature, a non-negative matrix factorization (NMF) model classified gastric cancer (GC) cases into subgroups with significantly different Disease-free survival (Kaplan-Meier test, P = 0.013). In contrast, the NMF model based on all miRNAs did not associate with cancer outcome. The mutp53 miRNA signature associated with the outcomes of breast cancer (P = 0.024) and hepatocellular cancer (P = 0.012). The miRPath analysis revealed that mutp53-suppressed miRNAs associate with Hippo, TGF-β and stem cell signaling pathways. Taken together, our results highlight a miRNA-mediated GOF mechanism of mutant p53 on Arg282, and suggest the prognostic potential of mutp53-associated miRNA signature.

Les porphyries héréditaires : anomalies du métabolisme de l’hème

The hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway characterised by acute neurovisceral symptoms, skin lesions or both. Each porphyria is caused by abnormal function of a separate enzymatic step resulting in a specific accumulation of haem precursors. Seven porphyrias are the consequence of a partial enzyme deficiency while a gain of function mechanism has been recently characterised in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation, confusion and seizure, which may be life threatening. Cutaneous porphyrias can be present with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still underdiagnosed, but once they are suspected, and depending on the clinical presentation, a specific and simple front line test allows the diagnosis in all symptomatic patients. Diagnosis is essential to institute as soon as possible a specific treatment. Screening families to identify presymptomatic carriers is crucial to prevent chronic complications and overt disease by counselling on avoiding potential precipitants.

Involvement of the Apoer2 and Lrp1 receptors in mediating the pathological effects of ApoE4 in vivo

This study investigated the possible role of the ApoE receptors Lrp1 and Apoer2 in mediating the pathological effects of ApoE4 in ApoE-targeted-replacement mice expressing either the human ApoE3 or ApoE4 allele. In this study we show that activation of the amyloid cascade by inhibition of the Aβ-degrading enzyme neprilysin results in upregulation of the ApoE receptor Lrp1 in the CA1 hippocampal neurons of 4-month-old ApoE4 mice, but not in the corresponding ApoE3 or ApoE-deficient (KO) mice. These results are in accordance with the previous findings that activation of the amyloid cascade induces Aβ accumulation in the CA1 neurons of ApoE4 mice, but not in ApoE3 or ApoE-KO mice. This suggests that the apoE4-driven elevation of Lrp1 is mediated via a gain of function mechanism and may play a role in mediating the effects of ApoE4 on Aβ. In contrast, no changes were observed in the levels of the corresponding Apoer2 receptor following the neprilysin inhibition. The ApoE receptors of naive ApoE4 mice were also affected differentially and isoform specifically by ApoE4. However, under these conditions, the effect was an ApoE4-driven reduction in the levels of Apoer2 in CA1 and CA3 pyramidal neurons, whereas the levels of Lrp1 were not affected. RT-PCR measurements revealed that the levels of Apoer2 and Lrp1 mRNA in the hippocampus of naïve and neprilysin-inhibited mice were not affected by ApoE4, suggesting that the observed effects of ApoE4 on the levels of these receptors is posttranscriptional. In conclusion, this study shows that the levels of hippocampal ApoE receptors Lrp1 and Apoer2 in vivo are affected isoform specifically by ApoE4 and that the type of receptor affected is context dependent.

Abstract A7: Targeting mutant p53 and cell-cell adhesion in ovarian cancer

Abstract One of the most important challenges in the ovarian cancer research is to identify new molecular pathways that can be targeted to kill ovarian cancer cells. High-grade serous ovarian cancer (HGS-OvCa) cells survive, proliferate, and assemble into multicellular clusters that have a capacity to disseminate within the peritoneum. Therefore, it is important to identify specific molecular mechanisms that can be targeted to block survival, growth, and assembly of ovarian cancer multicellular clusters that metastasize within peritoneum. High-grade ovarian tumors originate from the fallopian tube epithelium (FTE) and almost all have mutations in theTP53 gene. The contribution of various mutant p53 proteins (m-p53) to ovarian cancer and whether targeting mutant p53 –dependent molecular mechanisms in ovarian cancer is poorly understood and of clinical significance. We have developed new methodologies and used them to identify m-p53-dependent pathways that support HGS-OvCa survival, proliferation and assembly of multicellular aggregates under condition mimicking peritoneal suspension. To determine whether mutations in TP53 contribute to the ability of HGS-OvCa cells to survive, proliferate, and assemble into multicellular clusters we introduced different variants of mutant p53 (m-p53) into normal human non-transformed FTE cells. We found that expression of m-p53 variants (R175H; R249S; R273H) in FTE cells promoted survival, growth, and assembly of multicellular clusters under conditions mimicking peritoneal suspension. Interestingly m-p53-driven phenotypes were not induced by the loss of p53 expression; and more importantly, these phenotypes were blocked by the reactivation of p53 pathway with small molecule inhibitor NSC319726. We further found that m-p53 induced phenotypes were dependent on the expression of integrin β 1 (ITGB1). The evidence that activated ITGB1 is enriched at cell's surface of precursors of HGS-OvCa within the fallopian tube and HGS-OvCa isolated from ovarian cancer patients highlighted the relevance of these findings to ovarian cancer. We hypothesize that mutant p53 supports survival, growth, and assembly of multicellular HGS-OvCa clusters through mechanisms in which ITGB1 plays a critical role. Our data indicate that observed phenotypes are the consequence of the m-p53 gain of function mechanisms that are amenable to therapeutic inhibitions and targeting m-p53 in HGS-OvCa might be of clinical significance. Citation Format: Marcin P. Iwanicki, Marian Novak, Ioannis K. Zervantonakis, Tan A. Ince, Ronny Drapkin, Joan S. Brugge. Targeting mutant p53 and cell-cell adhesion in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A7.

191st ENMC International Workshop: Recent advances in oculopharyngeal muscular dystrophy research: From bench to bedside 8-10 June 2012, Naarden, The Netherlands

This workshop hosted a group of 19 clinicians and basic scientist from 5 countries (France, USA, Canada, Denmark and The Netherlands). Recent research developments and possible collaboration in advancing therapies for oculopharyngeal muscular dystrophy (OPMD), a late onset, rare and progressive neurodegeneration disorder were discussed in this meeting. The disease is caused by alanine expansion mutations in the gene encoding for poly(A)-binding protein nuclear 1 (PABPN1), resulting in accumulation of mutant PABPN1 in the cell nucleus and the formation of insoluble aggregates. Therefore, OPMD is categorized together with neurodegenerative disorders that are caused by single amino acid repeats. While the genetic cause for those disorders is often recognized, the molecular basis for symptoms is still obscure. PABPN1 is ubiquitously expressed and it is not known why symptoms develop only after midlife and initially only in subsets of skeletal muscles [1,2]. While the genetic cause for OPMD was reported in 1998 and good surgical treatments exist for major early symptoms, there are currently no medical options for the more general skeletal muscle involvement, no validated biomarkers and no clinical severity scale.

Comprehensive Clinical and Molecular Analysis of 12 Families with Type 1 Recessive Cutis Laxa

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFβ) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFβ activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

Progressive neuronal inclusion formation and axonal degeneration in CHMP2B mutant transgenic mice

Mutations in the charged multivesicular body protein 2B (CHMP2B) gene cause frontotemporal lobar degeneration. The mutations lead to C-terminal truncation of the CHMP2B protein. We generated Chmp2b knockout mice and transgenic mice expressing either wild-type or C-terminally truncated mutant CHMP2B. The transgenic CHMP2B mutant mice have decreased survival and show progressive neurodegenerative changes including gliosis and increasing accumulation of p62- and ubiquitin-positive inclusions. The inclusions are negative for the TAR DNA binding protein 43 and fused in sarcoma proteins, mimicking the inclusions observed in patients with CHMP2B mutation. Mice transgenic for mutant CHMP2B also develop an early and progressive axonopathy characterized by numerous amyloid precursor protein-positive axonal swellings, implicating altered axonal function in disease pathogenesis. These findings were not observed in Chmp2b knockout mice or in transgenic mice expressing wild-type CHMP2B, indicating that CHMP2B mutations induce degenerative changes through a gain of function mechanism. These data describe the first mouse model of dementia caused by CHMP2B mutation and provide new insights into the mechanisms of CHMP2B-induced neurodegeneration.

Transcriptome-Wide Analysis of Polyadenylation Sites in Mantle Cell Lymphoma Using Polyadenylation Signal Sequencing,

Abstract 3478In mantle cell lymphoma (MCL) cyclin D1 overexpression is caused by the translocation t(11;14) and mutations in the 3'UTR of cyclin D1 further enhance its expression. Notably, higher levels of the shorter cyclin D1 isoform correlate with inferior clinical outcome. Alternative polyadenylation (APA) is an important post-transcriptional mechanism that controls mRNA stability, subcellular localization, and protein translation. 3'UTR shortening has been associated with proliferation and oncogenic transformation.We analyzed the polyadenylome in 10 MCL cell lines, in CD19+ selected tumor cells from 2 leukemic and 1 nodal MCL sample, in core biopsies from 2 nodal MCL, and in peripheral blood B cells from 2 healthy donors by polyadenylation (signal) sequencing (PA-seq), which results in short sequencing reads comprising the polyadenylation (PA) site. Reads per sample were scaled to the median of total reads in order to normalize samples for cross comparison. We called a PA site if the sum of reads in a window of −2 to +25nt located in the 3’UTR contributed ≥ 10% of total reads for the gene in one sample, and ≥ 2 samples had ≥ 5 reads at a given location. A gene was considered to have multiple PA sites if > 1 site met the minimum criteria in > 1 sample. First, we identified 13,057 PA sites representing 7,115 genes mapped to the plus strand. Of these genes 25% had 2, 14% 3 and 9% ≥ 4 PA sites, respectively. Owing to the generally repressive nature of the 3’UTR, shortening of this region may give rise to a gain of function. We therefore sought to identify genes in which tumor cells display shorter mRNA transcripts than normal B cells. We identified 217 transcripts where the most proximal PA site of a gene was used by tumor but not normal cells. These genes belong to a wide range of different functional groups. Analyses into the possible role of these genes in MCL pathogenesis and into the mechanism contributing to APA are ongoing.3'UTR shortening of cyclin D1 transcripts in MCL as a mechanism for proto-oncogene activation has been previously reported. However, in a substantial number of cases in which short cyclin D1 transcripts were detected the mechanism of APA has remained elusive. We were therefore particularly interested in cyclin D1. Using the criteria described above we identified 3 PA sites, a proximal site previously identified in tumor cells, the consensus site, about 3.5kb distal to the first site and an additional, previously unreported site further 3' that was used in only one cell line. Five of 10 MCL cell lines and 2 of 5 primary tumor samples preferentially used the proximal PA site. Consistent with the role of the 3'UTR in destabilizing cyclin D1 mRNA, the 7 MCL samples with preferential use of the proximal site had on average 3-fold higher expression than those that preferentially used the distal site (p=0.02). The only exception is the Mino cell line that had the highest cyclin D1 expression across all samples despite preferential usage of the distal site. Moreover, we found that normal B cells also used the proximal PA site albeit with very low numbers of cyclin D1 transcripts overall. Next we compiled the sequences at the PA sites to test for mutations that may give rise to APA. The canonical AAUAAA hexamer represents the most frequent PA signal but hexamer variants have been described. Mutations in MCL and the MCL-derived cell line Z138 have been reported to create an aberrant proximal AAUAAA hexamer in cyclin D1 3'UTR. We identified the previously described A insertion in Z138 cells, but there was no mutation in any of the other samples. The absence of AAUAAA and variant sequences suggest that the pentamer sequence AAUAA may mediate APA under select circumstances. Why this site can be used by some but not other cells will be interesting to address and may reveal an additional layer of regulation.In summary, we have begun to systematically address the role of APA as a gain of function mechanism in MCL. Hundreds of genes give rise to shorter transcripts in MCL cells than in normal B cells, and it will be interesting to investigate whether these genes contribute to MCL pathogenesis. The analysis of APA in cyclin D1 suggests that under certain circumstances, the exact conditions remain to be defined, pentameric PA sequences may be used efficiently and can give rise to a gain of function genotype. Disclosures:Weniger:NHLBI, Intramural Research Program: Research Funding. Liu:NHLBI, Intramural Research Program: Research Funding. Ni:NHLBI, Intramural Research Program: Research Funding. Zhu:NHLBI, Intramural Research Program: Research Funding. Wiestner:NHLBI, Intramural Research Program: Research Funding.

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

X-linked Charcot-Marie-Tooth Disease (CMTX) in a Severely Affected Female Patient with Scattered Lesions in Cerebral White Matter

Charcot-Marie-Tooth neuropathy (CMT) is an inherited degenerative disorder of the peripheral nervous system that results in slowly progressive distal muscle weakness, atrophy and loss of proprioception in the affected areas. X-linked CMT (CMTX) has been localized to the pericentric region of the X chromosome. CMTX neuropathy is usually associated with mutations in exon 2 of the gap junction protein beta1 (GJB1) gene. GJB1 is a gap junction protein expressed in various cells including oligodendrocytes, astrocytes and myelinating schwann cells. Here, we report a female case of CMTX with a GJB1 mutation. The patient was severely clinically affected and exhibited both the features of demyelination and axonopathy. This is the first female patient with CMTX who showed permanent atypical scattered lesions in cerebral white matter of the brain on T2-weighted magnetic resonance images (MRI), which is very rare. The existence of a female patient with severe clinical symptoms may show that gain of function mechanism also leads to the disorders seen in these patients.

Autosomal Dominant Cutis Laxa with Severe Lung Disease: Synthesis and Matrix Deposition of Mutant Tropoelastin

Cutis laxa (CL) is a heterogeneous group of genetic and acquired disorders with at least two autosomal dominant forms caused by mutations in the elastin and fibulin-5 genes, respectively. To define the molecular basis of CL in patients negative for point mutations in the elastin gene, metabolic labeling and immunoprecipitation experiments were used to study the synthesis of elastin in dermal fibroblasts. In addition to the normal 68 kDa tropoelastin (TE) protein, an abnormal, 120 kDa polypeptide was detected in the proband and her affected daughter in a CL family characterized by hernias and unusually severe and early-onset pulmonary disease including bronchiectasis and pulmonary emphysema. Mutational and gene expression studies established that affected individuals in this family carried a partial tandem duplication in the elastin locus. Immunoprecipitation experiments showed that the mutant TE was partially secreted and partially retained intracellularly. A polyclonal antibody raised against a unique peptide in the mutant TE molecule showed both intracellular and matrix staining. We conclude that elastin mutations can cause CL associated with a severe pulmonary phenotype. Synthesis of abnormal TE may interfere with elastic fiber function through a dominant-negative or a gain of function mechanism.

Do oxidatively modified proteins cause ALS?

Over 90 individual mutations in SOD1 are known to cause familial amyotrophic lateral sclerosis (FALS). It is widely accepted that these mutations exert their toxic effects by a gain of function mechanism, but the nature of these toxic effects is as yet unknown. It has been proposed by several laboratories that reactions of FALS-mutant CuZnSOD are the source of elevated oxidative stress in CuZnSOD-linked FALS. It has also been proposed that aggregates of CuZnSOD are somehow involved in the disease. The hypothesis that aggregates of CuZnSOD cause ALS is particularly attractive because protein aggregates are frequently associated with other neurodegenerative diseases. Recent evidence increasingly suggests that protein aggregates containing CuZnSOD protein play a role in CuZnSOD-linked ALS, but it is not yet know why the aggregates form nor if the CuZnSOD proteins in the aggregates are cleaved, oxidized, demetallated, or otherwise covalently modified.

Integrity of the N-terminal transcription domain of p53 is required for mutant p53 interference with drug-induced apoptosis.

The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N-terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug-induced apoptosis, further modification at the N-terminus abolished this blocking activity. Furthermore, expression of c-myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53-expressing cells, but was abolished in the presence of the transcription-deficient p53 core mutant. In addition, wild-type p53, mutated in the N-terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N-terminus is essential for both the activity of wild-type p53 in apoptosis and for mutant p53-mediated block of drug-induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.