Aspergillus fumigatus conidial metalloprotease Mep1p cleaves host complement proteins.

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In†Vivo.

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.

Polymorphism of Amyloid Fibrils In Vivo.

Polymorphism is a wide-spread feature of amyloid-like fibrils formed in vitro, but it has so far remained unclear whether the fibrils formed within a patient are also affected by this phenomenon. In this study we show that the amyloid fibrils within a diseased individual can vary considerably in their three-dimensional architecture. We demonstrate this heterogeneity with amyloid fibrils deposited within different organs, formed from sequentially non-homologous polypeptide chains and affecting human or animals. Irrespective of amyloid type or source, we found in vivo fibrils to be polymorphic. These data imply that the chemical principles of fibril assembly that lead to such polymorphism are fundamentally conserved in vivo and in vitro.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex.

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B.

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

Loss of the protein-tyrosine phosphatase DEP-1/PTPRJ drives meningioma cell motility.

DEP-1/PTPRJ is a transmembrane protein-tyrosine phosphatase which has been proposed as a suppressor of epithelial tumors. We have found loss of heterozygosity (LOH) of the PTPRJ gene and loss of DEP-1 protein expression in a subset of human meningiomas. RNAi-mediated suppression of DEP-1 in DEP-1 positive meningioma cell lines caused enhanced motility and colony formation in semi-solid media. Cells devoid of DEP-1 exhibited enhanced signaling of endogenous platelet-derived growth factor (PDGF) receptors, and reduced paxillin phosphorylation upon seeding. Moreover, DEP-1 loss caused diminished adhesion to different matrices, and impaired cell spreading. DEP-1-deficient meningioma cells exhibited invasive growth in an orthotopic xenotransplantation model in nude mice, indicating that elevated motility translates into a biological phenotype in vivo. We propose that negative regulation of PDGF receptor signaling and positive regulation of adhesion signaling by DEP-1 cooperate in inhibition of meningioma cell motility, and possibly tumor invasiveness. These phenotypes of DEP-1 loss reveal functions of DEP-1 in adherent cells, and may be more generally relevant for tumorigenesis.

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.