Kreatinstoffwechsel in humaner Haut

Im Rahmen der vorliegenden Arbeit wurden verschiedene Aspekte des Kreatinmetabolismus in humaner Haut untersucht. Diese Aspekte waren im Einzelnen: Die zytosolische Kreatinkinase (CK-BB), die mitochondriale Kreatinkinase (CK-mt) sowie der Kreatintransporter (CT) konnten in humaner Haut immunhistologisch und mit Western-Blot-Analysen nachgewiesen werden. Ferner konnten die Lokalisation der CK-BB und CK-mt und die Funktionalität dieser Enzyme auch in vitro in primären, humanen Keratinozyten gezeigt werden. Zudem wurde die aktive Aufnahme von Kreatin in primären, humanen Keratinozyten und primären, humanen, dermalen Fibroblasten über den CT untersucht. Die Kreatinkinase-Aktivität wurde in primären, humanen Keratinozyten junger und alter Spender gemessen, wobei sich eine Reduktion der Aktivität im Verlauf des Alterungsprozesses zeigte. Dieser Reduktion konnte durch die Supplementation mit Kreatin ebenso entgegen gewirkt werden, wie der durch Wasserstoffperoxid verursachten Inhibition der Kreatinkinase-Aktivität. Somit konnte für Kreatin eine protektive Wirkung gegen oxidativen Stress, verursacht von vermehrt z.B. während des Alterungsprozesses auftretenden Reaktiven Sauerstoffspezies (ROS), gezeigt werden. Dieser Schutzeffekt kann durch die stabilisierende Wirkung von Kreatin auf CK-mt und damit auf die Mitochondrienmembran erklärt werden, da CK-mt die vorherrschende Isoform in Keratinozyten zu sein scheint. Die Schlüsselenzyme der Kreatinsynthese, Guanidinoacetat-Methyl-Trans-ferase (GAMT) und Arginin-Glycin-Amidino-Transferase (AGAT), konnten sowohl in humaner Haut als auch in primären, humanen Keratinozyten und primären, humanen, dermalen Fibroblasten auf mRNA Ebene nachgewiesen werden. Weiterhin zeigte sich bei Keratinozyten in vitro eine Stimulierbarkeit der Kreatinkinase-Aktivität nach Inkubation mit der Kreatinsynthesevorstufe Guanidinoacetat. Die Inhibition der CK-BB und CK-mt in Zellen der humanen Keratinozyten-Zelllinie HaCat und der humanen Gebärmutterhalskarzinom-Zelllinie HeLaS3 zeigt zum Teil drastische Auswirkungen auf die Zelle. Besonders die Inaktivierung der CK-mt führt zu deutlich zytotoxischen Effekten, verminderter Proliferation und einem Absinken des Mitochondrienmembranpotentials. Die Auswirkungen des Verlustes der CK-mt sind dabei auch elektronenmikroskopisch anhand von Veränderungen der Mitochon-drienmembranen zu sehen. In Bezug auf apoptotische und nekrotische Prozesse lässt sich jeweils bei Ausschalten der in dem Zelltyp geringer exprimierten Kreatinkinase der stärkere Anstieg beobachten. Die gewonnenen Daten zeigen nicht nur die Bedeutung der Kreatinkinasen für die Zelle auf, sondern unterstützen auch die Befunde zur besonderen Rolle der CK-mt bei der Stabilisierung der Mitochondrienmembran

Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in GDAP1 impede the protein’s role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it’s activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation.ISSN:2045-232

Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc–Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the α6 and β1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-β. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of β1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc–Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment

Specificity Protein 2 (Sp2) Is Essential for Mouse Development and Autonomous Proliferation of Mouse Embryonic Fibroblasts

BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo

Differential expression and sex chromosome association of CHD3/4 and CHD5 during spermatogenesis

ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation. Indeed, loss of CHD5 contributes to neuroblastoma. In this study, we first demonstrate that CHD5 is a nucleosome-stimulated ATPase. We then compare CHD3/4 and CHD5 expression in mouse brain and show that CHD5 expression is restricted to a subset of cortical and hippocampal neurons whereas CHD3/4 expression is more widespread. We also uncover high levels of CHD5 expression in testis. CHD5 is transiently expressed in differentiating germ cells. Expression is first detected in nuclei of postmeiotic round spermatids, reaches a maximum in stage VIII spermatids and then falls to undetectable levels in stage IX spermatids. Surprisingly, CHD3/4 and CHD5 show complementary expression patterns during spermatogenesis with CHD3/ 4 levels progressively decreasing as CHD5 expression increases. In spermatocytes, CHD3/4 localizes to the pseudoautosomal region, the X centromeric region and then spreads into the XY body chromatin. In postmeiotic cells, CHD5 colocalises with macroH2A1.2 in association with centromeres and part of the Y chromosome. The subnuclear localisations of CHD4 and CHD5 suggest specif

Specificity protein 2 (Sp2) is essential for mouse development and autonomous proliferation of mouse embryonic fibroblasts

Background: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. Methodology/Principal Findings: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. Conclusions/Significance: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with th

Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.(VLID)276376

Miz1 Is a Critical Repressor of cdkn1a during Skin Tumorigenesis

The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15Ink4), cdkn1a (p21Cip1), and cdkn1c (p57Kip2). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin

Schwann cell Myc-interacting zinc-finger protein 1 without pox virus and zinc finger: epigenetic implications in a peripheral neuropathy

Functionality of adult peripheral nerves essentially relies on differentiation of Schwann cells during postnatal development, as well as fine-tuned re- and transdifferentiation in response to peripheral nerve injury. Epigenetic histone modifications play a major role during the differentiation of embryonic stem cells and diverse organ specific progenitor cells, yet only little is known about the epigenetic regulation of Schwann cells. Just recently, Fuhrmann et al. reported how the transcription factor Myc-interacting zinc-finger protein 1 (Miz1) might contribute to Schwann cell differentiation through repression of the histone demethylase Kdm8. Here, we discuss the potential novel role of Miz1 in Schwann cell differentiation and give a short overview about previously reported histone modifications underlying peripheral nerve development and response to injury