Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy

Solid state NMR/Biophysical Organic Chemistr

Functional analysis of the murine 66.3-kDa protein

Im Mittelpunkt dieser Arbeit stand das 2005 erstmals beschriebene murine lysosomale Matrixprotein 66.3-kDa-Protein. Die Interaktion des 66.3-kDa-Proteins mit der lysosomalen Aspartylprotease Cathepsin D, welche während der ausführlichen molekularen Charakterisierung vermutet wurde, konnte im Rahmen der vorliegenden Arbeit mit verschiedenen Interaktionsstudien (Ko-Immunpräzipitation, Pepstatin-A-Chromatographie, Vernetzung mit heterobifunktionalen Quervernetzern) bestätigt werden. Das zuvor beschriebene Prozessierungsmuster für das 66.3-kDa-Protein konnte durch die Identifizierung eines weiteren Fragments mittels MALDI-TOF-MS/PMF und Western-Blot-Untersuchungen vervollständigt werden. Die Prozessierung des 66.3-kDa-Proteins stellt sich demnach als zweistufiger autokatalytischer Prozess dar. Die drei entstandenen Fragmente bleiben nicht-kovalent miteinander verknüpft und stellen vermutlich die aktive Form des 66.3-kDa-Proteins dar. Zudem konnte mittels Gelfiltration und Querv! ernetzung gezeigt werden, dass je zwei 66.3-kDa-Proteine als funktionell stabiler Homodimer aus jeweils 2-3 Untereinheiten vorliegen. Durch Kristallisation des 66.3-kDa-Proteins konnten Röntgenbeugungsmuster generiert werden, mit deren Hilfe die dreidimensionale Struktur des Proteins aufgelöst wurde. Der Vergleich der dreidimensionalen Struktur des 66.3-kDa-Proteins mit bereits bekannten Proteinstrukturen konnte auf Grund der charakteristischen Anordnung der Peptidkette in einer αββα-Konformation die Zugehörigkeit des 66.3-kDa-Proteins in die Superfamilie der Ntn-Hydrolasen (Enzymklasse 3.5.1) zeigen und eine hydrolytische Funktion im Lipid-Stoffwechsel bei der Spaltung von linearen, nicht-peptidischen Amidbindungen vorhersagen. Mögliche Substrate für das 66.3-kDa-Protein wären somit u. a. verschiedene N-Acylethanolamide oder Sphingosine, sowie hydrophobe Proteinmodifikationen mit Amidbindungen wie die N-Myristoylierung, Glypiation oder Lysin-Acetylierung

Cathepsin D Polymorphism C224T in Childhood-Onset Neurodegenerative Disorders: No Impact for Childhood Dementia

Compromised lysosomal functioning has been identified as a major risk factor for neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Furthermore, the association between a defined cathepsin D ( CTSD ) polymorphism and a higher risk of sporadic Alzheimer's disease has been established for particular populations. Here, we analyzed 189 children with rare neurodegenerative disease for carrying the T-allele by polymerase chain reaction-restriction fragment length polymorphism. We found no statistical differences in genotype and allele frequencies between the neurodegenerative group and European descent participants of genetic studies using the Cochran-Armitage's trend test. In contrast to adult-onset neurodegenerative diseases, analysis of clinical datasets of children carrying the T-allele did not demonstrate differences to the general disease group

LC-MS Based Platform Simplifies Access to Metabolomics for Peroxisomal Disorders

Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit

Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography

<p>Abstract</p> <p>Background</p> <p>The lysosomal 66.3 kDa protein from mouse is a soluble, mannose 6-phosphate containing protein of so far unknown function. It is synthesized as a glycosylated 75 kDa precursor that undergoes limited proteolysis leading to a 28 kDa N- and a 40 kDa C-terminal fragment.</p> <p>Results</p> <p>In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core αββα sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily.</p> <p>Conclusion</p> <p>Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.</p

Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography

Lakomek K, Dickmanns A, Kettwig M, Urlaub H, Ficner R, Lübke T. Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography. BMC Structural Biology. 2009;9(1):56-72

Kidney Injury by Variants in the <i>COL4A5</i> Gene Aggravated by Polymorphisms in Slit Diaphragm Genes Causes Focal Segmental Glomerulosclerosis

Kidney injury due to focal segmental glomerulosclerosis (FSGS) is the most common primary glomerular disorder causing end-stage renal disease. Homozygous mutations in either glomerular basement membrane or slit diaphragm genes cause early renal failure. Heterozygous carriers develop renal symptoms late, if at all. In contrast to mutations in slit diaphragm genes, hetero- or hemizygous mutations in the X-chromosomal COL4A5 Alport gene have not yet been recognized as a major cause of kidney injury by FSGS. We identified cases of FSGS that were unexpectedly diagnosed: In addition to mutations in the X-chromosomal COL4A5 type IV collagen gene, nephrin and podocin polymorphisms aggravated kidney damage, leading to FSGS with ruptures of the basement membrane in a toddler and early renal failure in heterozygous girls. The results of our case series study suggest a synergistic role for genes encoding basement membrane and slit diaphragm proteins as a cause of kidney injury due to FSGS. Our results demonstrate that the molecular genetics of different players in the glomerular filtration barrier can be used to evaluate causes of kidney injury. Given the high frequency of X-chromosomal carriers of Alport genes, the analysis of genes involved in the organization of podocyte architecture, the glomerular basement membrane, and the slit diaphragm will further improve our understanding of the pathogenesis of FSGS and guide prognosis of and therapy for hereditary glomerular kidney diseases