Structural insights into the MMACHC-MMADHC protein complex involved in vitamin B12 trafficking

Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations

ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFN gamma, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response

Human alpha-aminoadipic semialdehyde synthase (AASS); A Target Enabling Package

This work provides the early tools to develop substrate reduction inhibitors for a genetic childhood seizure disorder, with the hypothesis to target the enzyme (AASS) upstream of the defective gene (ALDH7A1) in the human lysine metabolic pathway. This TEP package includes recombinant human AASS purification protocols, structures of the AASS second domain in different states, in vitro assays to detect ligand binding (differential scanning fluorimetry) and enzyme activity (NADH formation) of human AASS, as well as initial chemical matters. A description of the materials and methods is included within the TEP datasheet

Human alpha-aminoadipic semialdehyde synthase (AASS); A Target Enabling Package

This work provides the early tools to develop substrate reduction inhibitors for a genetic childhood seizure disorder, with the hypothesis to target the enzyme (AASS) upstream of the defective gene (ALDH7A1) in the human lysine metabolic pathway. This TEP package includes recombinant human AASS purification protocols, structures of the AASS second domain in different states, in vitro assays to detect ligand binding (differential scanning fluorimetry) and enzyme activity (NADH formation) of human AASS, as well as initial chemical matters

Strukturanalyse von DNA-Relaxasen, den Schlüsselenzymen der bakteriellen Konjugation: TraA und die N-terminale Relaxase Domäne aus Gram-positiven Plasmid pIP501 zeigen spezifische Bindung an oriT und bilden Dimere in Lösung

Die DNA Relaxase TraA ist auf dem konjugativen Plasmid pIP501, das einen breiten Wirtsbereich aufweist, kodiert. Es ist die zweite charakterisierte Relaxase, die aus einem Gram positiven Organismus stammt, nach MobMpMV158. TraA (654 Aminosäuren) und die N-terminale Domäne (Aminosäuren 1-246), bezeichnet als TraAN246, wurden als His-tag Fusionsproteine exprimiert und gereinigt. Kleinwinkelröntgenstreuung und chemische Quervernetzung bewiesen, dass TraA und TraAN246 Dimere in Lösung bilden. Beide Proteine zeigten in vitro oriTpIP501 Spaltungsaktivität für Superhelix DNA. oriT Bindung der Relaxase wurde mittels Gel Retardations Assay nachgewiesen. Radioaktiv markierte Oligonukleotide, die verschiedene Teile von oriTpIP501 umfassten, wurden zur Bindung mit TraA und TraAN246 eingesetzt. Das längste Target Oligonukleotid enthält eine Haarnadelstruktur, die Nickstelle und 7 weitere Nukleotide. Die Dissoziationskonstante des Protein-DNA Komplexes beträgt 55 nM für TraA und 26 nM für TraAN246. Die Entfaltung von beiden Proteinkonstrukten wurde mittels Zirkulärem Dichroismus (CD) analysiert. Die Schmelztemperatur (TM), detektiert durch Signaländerung bei 220 nm, wurde für beide Proteine als 42°C bestimmt. Die CD Spektren, gemessen bei 20°C, zeigten für TraA 30 % -helikale und 13 % -Faltblatt Strukturen und für das verkürzte Protein 27 % -helikale und 18 % - Faltblatt Strukturen. Die Tatsache, dass die DNA Bindung Sekundärstrukturgehalt und thermische Stabilität von TraAN246 erhöht, weist auf einen induzierten Fit Mechanismus für die Entstehung von spezifischen Relaxase-oriT Komplexen hin. Beide Proteine binden die Promotor Region des tra Operons und regulieren dadurch seine Transkription. Weiters wurde im Rahmen dieser Arbeit die von pIP501 kodierte lytische Transglykosylase (LT) Orf7 untersucht. Die Expression und Reinigung von löslichem Orf7 Protein aus E. coli wurde etabliert. 3D Strukturmodelle wurden für Orf10pIP501 und die LT Domäne von Orf7 berechnet. Diese Modelle können als Suchmodelle zur Lösung der Kristallstruktur mittels der molekularen Ersatzmethode (molecular replacement) benutzt werden.TraA is the DNA relaxase encoded by the broad-host-range Gram-positive plasmid pIP501. It is the second relaxase characterized from plasmids originating from Gram-positive organisms. TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabeled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein-DNA complex encompassing the inverted repeat (IR), the nick site and additional 7 bases, was found to be 55 nM for TraA, and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the circular dichroism (CD) signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at around 42°C. CD spectra measured at 20°C showed 30 % α-helix and 13 % β-sheet for TraA and 27 % α-helix and 18 % β-sheet content for the truncated protein. Upon DNA binding an enhanced secondary structure content and increased thermal stability was observed for the TraAN246 protein suggesting an induced-fit mechanism for the formation of the specific relaxase-oriT complex. Both proteins bind to promoter region of tra operon and therefore regulate its transcription. Expression of soluble Orf7 protein was achieved, the protein turned out to possess lytic transglycosylase (LT) activity. 3D structure models were calculated for Orf10 and LT domain of Orf7. These models can be used as search models in crystal structure solution by molecular replacement method