Uschi Lindert

The taste of heavy metals: Gene regulation by MTF-1☆

The metal-responsive transcription factor-1 (MTF-1, also termed MRE-binding transcription factor-1 or metal regulatory transcription factor-1) is a pluripotent transcriptional regulator involved in cellular adaptation to various stress conditions, primarily exposure to heavy metals but also to hypoxia or oxidative stress. MTF-1 is evolutionarily conserved from insects to humans and is the main activator of metallothionein genes, which encode small cysteine-rich proteins that can scavenge toxic heavy metals and free radicals. MTF-1 has been suggested to act as an intracellular metal sensor but evidence for direct metal sensing was scarce. Here we review recent advances in our understanding of MTF-1 regulation with a focus on the mechanism underlying heavy metal responsiveness and transcriptional activation mediated by mammalian or Drosophila MTF-1. This article is part of a Special Issue entitled: Cell Biology of Metals.

Identification of a Mutation Causing Deficient BMP1/mTLD Proteolytic Activity in Autosomal Recessive Osteogenesis Imperfecta

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR‐OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR‐OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C‐terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient‐derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C‐terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR‐OI. Hum Mutat 33:343–350, 2012.

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

Metal-responsive transcription factor 1 (MTF-1) activity is regulated by a nonconventional nuclear localization signal and a metal-responsive transactivation domain.

ABSTRACT Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; “humanizing” mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

Genotype–phenotype study in type V osteogenesis imperfecta

Type V osteogenesis imperfecta (OI) presents with moderate-to-severe skeletal deformity and is characterized by hyperplastic callus formation at fracture sites and calcification of the interosseous membranes of the forearm and lower leg. The facial dysmorphism is not well characterized and has not been described in previous reports. Inheritance is autosomal dominant, although the genetic aetiology remained unknown until very recently. The aims of this study were to establish the genetic aetiology in patients with type V OI and further characterize patients with this condition, and to ascertain whether they have a similar clinical phenotype and facial dysmorphism. Three families (one mother–daughter pair and two singletons) were identified with the above features and further investigations (molecular genetic analysis and skin biopsy including electron microscopy, histology and collagen species analysis) were performed. Accurate phenotyping of patients with type V OI was performed. PCR amplification was performed using the Sheffield Diagnostic Genetics Service pyrosequencing assay for the interferon-induced transmembrane protein-5 (IFITM5) gene. All the patients had been confirmed to have a heterozygous variant, c.[−14C>T];[=], in the 5′-UTR of the IFITM5 gene, which is located in the transcribed region of this gene. This recurrent mutation, in IFITM5, also known as bone-restricted interferon-induced transmembrane protein-like protein or BRIL, encodes a protein with a function in bone formation and plays an important role in osteoblast formation. All four patients in this study appear to have similar clinical features and facial dysmorphism, including a short, up-turned nose, a small mouth, a prominent chin and greyish-blue sclerae. Skin biopsy in one patient showed clumping of elastic fibres and normal biochemical analysis of collagen. We have been able to characterize patients with type V OI further and confirm the genetic aetiology in this distinct form of OI. Accurate phenotyping (including describing the common facial features) before investigations is important to enable the useful interpretation of genetic results and/or target-specific gene testing.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

Background: The collagen chaperone HSP47 is implicated in recessive osteogenesis imperfecta (OI). Results: In OI dachshunds, an HSP47(L326P) mutation affects the post-translational modification, secretion, and cross-linking of collagen type I. Conclusion: Impaired chaperone function, ER stress, and aberrant bone collagen cross-linking are implicated in the disease mechanism. Significance: Our findings are relevant for the diagnosis and pathological understanding of OI caused by an HSP47 defect. Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Molecular Consequences of Defective SERPINH1/HSP47 in the Dachshund Natural Model of Osteogenesis Imperfecta

Background: The collagen chaperone HSP47 is implicated in recessive osteogenesis imperfecta (OI). Results: In OI dachshunds, an HSP47(L326P) mutation affects the post-translational modification, secretion, and cross-linking of collagen type I. Conclusion: Impaired chaperone function, ER stress, and aberrant bone collagen cross-linking are implicated in the disease mechanism. Significance: Our findings are relevant for the diagnosis and pathological understanding of OI caused by an HSP47 defect. Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Characterization of metal-responsive transcription factor (MTF-1) from the giant rodent capybara reveals features in common with human as well as with small rodents (mouse, rat). Short communication.

From mammals to insects, metal‐responsive transcription factor 1 (MTF‐1) is essential for the activation of metallothionein genes upon heavy‐metal load. We have previously found that human MTF‐1 induces a stronger metal response than mouse MTF‐1. The latter differs from the human one in a number of amino acid positions and is also shorter by 78 aa at its C‐terminus. We reasoned that the weaker metal inducibility might be associated with a lesser demand for tight metal homeostasis in a low‐weight, short‐lived animal, and thus set out to determine the sequence of MTF‐1 from the largest living rodent, the Brazilian capybara that can reach 65 kg and also has a considerably longer life span than smaller rodents. An expression clone for capybara MTF‐1 was then tested for its activity in both mouse and human cells. Our analysis revealed three unexpected features: i) capybara MTF‐1 in terms of amino acid sequence is much more closely related to human than to mouse MTF‐1, suggesting an accelerated evolution of MTF‐1 in the evolutionary branch leading to small rodents; ii) capybara MTF‐1 is even 32 aa shorter at its C‐terminus than mouse MTF‐1, and iii) in an activity test, it is not more active than mouse MTF‐1. The latter two findings might indicate that capybara has evolved in an environment with low heavy‐metal load.

Expanding the clinical and mutational spectrum of the Ehlers-Danlos syndrome, dermatosparaxis type.

Purpose:The Ehlers–Danlos syndrome (EDS), dermatosparaxis type, is a recessively inherited connective tissue disorder caused by deficient activity of ADAMTS-2, an enzyme that cleaves the aminoterminal propeptide domain of types I, II, and III procollagen. Only 10 EDS dermatosparaxis patients have been reported, all presenting a recognizable phenotype with characteristic facial gestalt, extreme skin fragility and laxity, excessive bruising, and sometimes major complications due to visceral and vascular fragility.Methods:We report on five new EDS dermatosparaxis patients and provide a comprehensive overview of the current knowledge of the natural history of this condition.Results:We identified three novel homozygous loss-of-function mutations (c.2927_2928delCT, p.(Pro976Argfs*42); c.669_670dupG, p.(Pro224Argfs*24); and c.2751-2A>T) and one compound heterozygous mutation (c.2T>C, p.? and c.884_887delTGAA, p.(Met295Thrfs26*)) in ADAMTS2 in five patients from four unrelated families. Three of these displayed a phenotype that was strikingly milder than that of previously reported patients.Conclusion:This study expands the clinical and molecular spectrum of the dermatosparaxis type of EDS to include a milder phenotypic variant and stresses the importance of good clinical criteria. To address this, we propose an updated set of criteria that accurately captures the multisystemic nature of the dermatosparaxis type of EDS.Genet Med 18 9, 882–891.

Simian Virus 40 Strains with Novel Properties Generated by Replacing the Viral Enhancer with Synthetic Oligonucleotides

ABSTRACT Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an “enhancer trap” since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.