Inhibition of UV-induced matrix metabolism by a myristoyl tetrapeptide.

Regulation of extracellular matrix (ECM) composition is important in tissue homeostasis and function. We screened small peptides for their ability to inhibit ultraviolet (UV)-induced cell metabolism in epidermal fibroblasts. We found that UV irradiation increased matrix metalloproteinase (MMP) expression and inflammatory gene expression in human Hs68 fibroblast cells. We also demonstrated that a myristoyl tetrapeptide with the amino acid sequence Gly-Leu-Phe-Trp (mGLFW) suppressed the UV-induced expression of MMPs and inflammatory genes. Moreover, mGLFW stimulated the expression of ECM proteins in Hs68 fibroblasts. In order to provide the mechanism of action for mGLFW, we investigated UV-induced signaling changes in the presence of mGLFW using a cDNA microarray. UV exposure increased the expression of MMP genes, such as MMP1, MMP3, and MMP14, and inflammation-related genes, including interleukin 1 receptor and peroxisome proliferator-activated receptor gamma (PPARγ). Treatment with mGLFW abrogated the UV-induced expression of MMP-related genes and inflammatory genes. In addition, mGLFW increased the expression of collagen genes, including COL1A1, COL1A2, and COL5A1. We examined whether the activation of AP-1, a UV-activated transcription factor, is suppressed by mGLFW. The results demonstrated that AP-1 expression increased upon UV exposure and that this expression was inhibited by mGLFW. In conclusion, our results demonstrate that mGLFW reversed the effects of UV exposure by enhancing the expression of collagen proteins and suppressing the expression of MMPs, which degrade the ECM.

Smad-induced alterations of matrix metabolism by a myristoyl tetra peptide.

Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala-Ala-Pro-Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP-related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)-ß, indicating that the TGF-ß signaling pathway is crucial for simultaneous activation of several ECM-related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF-ß receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM-protecting agent.

Molecular bases of hearing loss in multi-systemic mitochondrial cytopathy.

PURPOSE: Hearing loss is a common clinical feature in classic mitochondrial syndromes. The purpose of this study was to evaluate the diverse molecular etiologies and natural history of hearing loss in multi-systemic mitochondrial cytopathies and the possible correlation between degree of hearing loss and neurological phenotype. METHODS: In this retrospective study we evaluated the clinical features and molecular bases of hearing loss associated with multi-systemic mitochondrial cytopathy. Forty-five patients with sensorineural hearing loss and definite diagnosis of mitochondrial cytopathy according to the published diagnostic criteria were studied. RESULTS: The sensorineural hearing loss was progressive and for the most part symmetrical with involvement of the higher frequencies. Both cochlear and retrocochlear involvement were found in this cohort. No correlation was found between the degree of hearing loss and the number and severity of neurological manifestations. Deleterious mtDNA point mutations of undisputed pathogenicity were identified in 18 patients. The A3243G mutation was the most frequently encountered among this group. MtDNA depletion, over-replication, and multiple deletions were found in further 11 cases. CONCLUSION: This study reveals an expanding spectrum of mtDNA abnormalities associated with hearing loss. No correlation was found between the degrees of hearing loss and the severity of neurological manifestations.

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness.

Patients with mitochondrial disease usually manifest multisystemic dysfunction with a broad clinical spectrum. When the tests for common mitochondrial DNA (mtDNA) point mutations are negative and the mtDNA defects are still hypothesized, it is necessary to screen the entire mitochondrial genome for unknown mutations in order to confirm the diagnosis. We report an 8-year-old girl who had a long history of ragged-red fiber myopathy, short stature, and deafness, who ultimately developed renal failure and fatal cardiac dysfunction. Respiratory chain enzyme analysis on muscle biopsy revealed deficiency in complexes I, II/III, and IV. Whole mitochondrial genome sequencing analysis was performed. Three novel changes: homoplasmic 15458T > C and 15519T > C in cytochrome b, and a near homoplasmic 5783G > A in tRNA(cys), were found in the proband in various tissues. Her mother and asymptomatic sibling also carry the two homoplasmic mutations and the heteroplasmic 5783G > A mutation in blood, hair follicles, and buccal cells, at lower percentage. The 5783G > A mutation occurs at the T arm of tRNA(cys), resulting in the disruption of the stem structure, which may reduce the stability of the tRNA. 15458T > C changes an amino acid serine to proline at a conserved alpha-helix, which may force the helix to bend. These two mutations may have pathogenic significance. This case emphasizes the importance of pursuing more extensive mutational analysis of mtDNA in the absence of common mtDNA point mutations or large deletions, when there is a high suspicion of a mitochondrial disorder.

Hearing loss in mitochondrial disorders.

Hearing loss is a common clinical feature in mitochondria-syndrome disorders. The underlining molecular etiology of hearing loss has not been fully investigated. In this study, 83 patients with mitochondrial syndromic hearing loss were evaluated clinically and their blood and tissue samples were examined molecularly. Using modified Walker's criteria, 31, 31, 14, and 7 patients had been classified as having definite, probable, possible, and unlikely diagnosis of mitochondrial disease, respectively. Deleterious mtDNA point mutations and/or abnormal mtDNA content or multiple deletions were identified in 20 patients with definite diagnosis and 2 patients with probable diagnosis. In addition to known, undisputed pathogenic mutations, several novel mutations believed to be clinically significant were found. Furthermore, abnormal mtDNA content and mtDNA deletions were found in some of the cases. Evaluation of clinical and diagnostic features associated with hearing loss revealed that cardiomyopathy, lactic acidosis, deficient respiratory chain enzyme complex activities, histochemical and ultrastructural abnormalities in mitochondria, and abnormal brain imaging results occurred significantly more frequently in patients with mtDNA alterations than in those without. This study revealed that the majority of the mtDNA defects in patients with mitochondrial syndromic hearing loss affect the overall mitochondrial gene expression.

Enhanced detection of deleterious mutations by TTGE analysis of mother and child's DNA side by side.

Mitochondrial DNA (mtDNA) disorders represent a group of heterogeneous diseases that are caused by mutations in mtDNA. We examined 45 pairs of mother and the affected child, by screening the entire mitochondrial genome with temporal temperature gradient gel electrophoresis (TTGE), using 32 pairs of overlapping primers. TTGE is an effective method of mutation detection. It detects and distinguishes heteroplasmic mutations from homoplasmic mutations. By running the mother and child's DNA samples side by side and sequencing only the DNA fragments showing different TTGE patterns, excessive sequencing can be avoided, particularly because most sequence variations represent benign polymorphisms. Mutations identified by sequencing were further confirmed by PCR/ASO (allele-specific oligonucleotide) dot blot analysis or PCR/RFLP (restriction fragment length polymorphism). A total of seven differences in sequence between mother and child pairs were identified: A189G, T5580C, G5821A, C5840T, A8326G, G12207A, and G15995A. All but two mutations were novel. The most significant are the A8326G, G12207A, and G15595A mutations. The A8326G is located at the anticodon region of tRNA(Lys), right next to the first nucleotide of the triplet codon, and it is highly conserved throughout evolution. The G12207A mutation is located at the first base of tRNA(ser) (AGY). The G15995A mutation occurs at a stem region that results in the disruption of the first base pair at the anticodon loop of tRNA(Pro) and is highly conserved throughout evolution from sea urchins to mammals. Running TTGE side by side with DNAs from mother and the affected child is a novel method to detect deleterious mutations.

A cystic fibrosis patient with two novel mutations in mitochondrial DNA: mild disease led to delayed diagnosis of both disorders.

A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for unknown mutations in the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE). Her asymptomatic mother's blood DNA was also analyzed and used as a reference. Two tRNA regions showing different TTGE patterns between the proband and her mother were sequenced. Two novel mutations, G15995A in tRNA(pro) and A8326G in tRNA(lys), were revealed. These mutations are present in heteroplasmic states. They both occurred at a nucleotide position that is highly conserved throughout evolution. This patient is also a compound heterozygote for the cystic fibrosis (CF) mutations, DeltaF508 and R347P. The phenotype for R347P has been associated with mild disease. Due to the mild features of the R347P mutation in the CF transmembrane conductance regulator (CFTR) gene and the heterogeneous clinical presentation of the mtDNA disease, the patient was not definitively diagnosed until age 21. This case underscores the importance of a complete mutational analysis of the entire mitochondrial genome when a patient suspected of mitochondrial disorder is negative for common mtDNA mutations.

Comprehensive scanning of the entire mitochondrial genome for mutations.

BACKGROUND: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. METHODS: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. RESULTS: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. CONCLUSIONS: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.

QM, a putative tumor suppressor, regulates proto-oncogene c-yes.

The QM gene encodes a 24.5 kDa ribosomal protein L10 known to be highly homologous to a Jun-binding protein (Jif-1), which inhibits the formation of Jun-Jun dimers. Here we have carried out screening with the c-Yes protein and found that a QM homologous protein showed interactions with c-Yes and other Src family members. We have found that two different regions of QM protein were associated with the SH3 domain of c-Yes. The QM protein does not contain canonical SH3 binding motifs or previously reported amino acid fragments showing interaction with SH3 domains. Several c-Yes kinase activity assays indicated that the QM protein reduced c-Yes kinase activity by 70% and that this suppression is related not only to the two SH3 binding regions but also to the C-terminal region of QM. Moreover, our autophosphorylation assays clarified that this regulation resulted from the inhibition of c-Yes autophosphorylation. Immunofluorescence studies showed that the QM proteins and c-Yes are able to interact in various tumor cell lines in vivo. The increases of the c-Yes protein and mRNA levels were detected when the QM was transfected. These results suggest that the QM protein might be a regulator for various signal transduction pathways involving SH3 domain-containing membrane proteins.