Does Generic Cyclic Kinase Insert Domain of Receptor Tyrosine Kinase KIT Clone Its Native Homologue?

Receptor tyrosine kinases (RTKs) are modular membrane proteins possessing both well-folded and disordered domains acting together in ligand-induced activation and regulation of post-transduction processes that tightly couple extracellular and cytoplasmic events. They ensure the fine-turning control of signal transmission by signal transduction. Deregulation of RTK KIT, including overexpression and gain of function mutations, has been detected in several human cancers. In this paper, we analysed by in silico techniques the Kinase Insert Domain (KID), a key platform of KIT transduction processes, as a generic macrocycle (KIDGC), a cleaved isolated polypeptide (KIDC), and a natively fused TKD domain (KIDD). We assumed that these KID species have similar structural and dynamic characteristics indicating the intrinsically disordered nature of this domain. This finding means that both polypeptides, cyclic KIDGC and linear KIDC, are valid models of KID integrated into the RTK KIT and will be helpful for further computational and empirical studies of post-transduction KIT events.

Human Vitamin K Epoxide Reductase as a Target of Its Redox Protein.

Human vitamin K epoxide reductase (hVKORC1) enzymatic activity requires an initial activation by a specific redox protein, a less studied step in the hVKORC1 vital cycle. Significant steric conditions must be met by enzymes, being that to adapt their configurations is mandatory for hVKORC1 activation. We studied, by molecular dynamics (MD) simulations, the folding and conformational plasticity of hVKORC1 in its inactive (fully oxidised) state using available structures, crystallographic and from de novo modelling. According to the obtained results, hVKORC1 is a modular protein composed of the stable transmembrane domain (TMD) and intrinsically disordered luminal (L) loop, possessing the great plasticity/adaptability required to perform various steps of the activation process. The docking (HADDOCK) of Protein Disulfide Isomerase (PDI) onto different hVKORC1 conformations clearly indicated that the most interpretable solutions were found on the target closed L-loop form, a prevalent conformation of hVKORC1's oxidised state. We also suggest that the cleaved L-loop is an appropriate entity to study hVKORC1 recognition/activation by its redox protein. Additionally, the application of hVKORC1 (membrane protein) in aqueous solution is likely to prove to be very useful in practice in either in silico studies or in vitro experiments.

The Inherent Coupling of Intrinsically Disordered Regions in the Multidomain Receptor Tyrosine Kinase KIT.

RTK KIT regulates a variety of crucial cellular processes via its cytoplasmic domain (CD), which is composed of the tyrosine kinase domain, crowned by the highly flexible domains-the juxtamembrane region, kinase insertion domain, and C-tail, which are key recruitment regions for downstream signalling proteins. To prepare a structural basis for the characterization of the interactions of KIT with its signalling proteins (KIT INTERACTOME), we generated the 3D model of the full-length CD attached to the transmembrane helix. This generic model of KIT in inactive state was studied by molecular dynamics simulation under conditions mimicking the natural environment of KIT. With the accurate atomistic description of the multidomain KIT dynamics, we explained its intrinsic (intra-domain) and extrinsic (inter-domain) disorder and represented the conformational assemble of KIT through free energy landscapes. Strongly coupled movements within each domain and between distant domains of KIT prove the functional interdependence of these regions, described as allosteric regulation, a phenomenon widely observed in many proteins. We suggested that KIT, in its inactive state, encodes all properties of the active protein and its post-transduction events.

Folding and Intrinsic Disorder of the Receptor Tyrosine Kinase KIT Insert Domain Seen by Conventional Molecular Dynamics Simulations.

The kinase insert domain (KID) of RTK KIT is the key recruitment region for downstream signalling proteins. KID, studied by molecular dynamics simulations as a cleaved polypeptide and as a native domain fused to KIT, showed intrinsic disorder represented by a set of heterogeneous conformations. The accurate atomistic models showed that the helical fold of KID is mainly sequence dependent. However, the reduced fold of the native KID suggests that its folding is allosterically controlled by the kinase domain. The tertiary structure of KID represents a compact array of highly variable α- and 310-helices linked by flexible loops playing a principal role in the conformational diversity. The helically folded KID retains a collapsed globule-like shape due to non-covalent interactions associated in a ternary hydrophobic core. The free energy landscapes constructed from first principles-the size, the measure of the average distance between the conformations, the amount of helices and the solvent-accessible surface area-describe the KID disorder through a collection of minima (wells), providing a direct evaluation of conformational ensembles. We found that the cleaved KID simulated with restricted N- and C-ends better reproduces the native KID than the isolated polypeptide. We suggest that a cyclic, generic KID would be best suited for future studies of KID f post-transduction effects.

Identification of the Primary Factors Determining theSpecificity of Human VKORC1 Recognition by Thioredoxin-Fold Proteins.

Redox (reduction-oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol-disulphide exchange reactions between PDI and hVKORC1.

Rheumatic disease presenting as septic arthritis: a report of 10 cases.

To determine the forms and characteristics of rheumatic diseases whose initial presentation mimics septic arthritis. Retrospective study of 398 patients hospitalized between 1979 and 2005 for arthritis diagnosed and treated as septic. In 10 cases, initial presentation of a rheumatic disease was highly suggestive of septic arthritis, and the patient was treated as such. Three had rheumatoid arthritis, 3 spondyloarthropathies, 2 unclassified rheumatic diseases, 1 Wegener granulomatosis and 1 cytosteatonecrosis. Mean time to diagnosis of rheumatic arthritis was 6 months. There were 7 males and 3 females aged from 15 to 77 years. Six had fever, and 3 had leucocytosis. Average ESR was 68 mm/1 h, and C-reactive protein was above 100 mg/l in 6 patients. Five patients had radiological signs suggestive of septic arthritis. Joint fluid count was above 100,000 WBCs/mm(3) in 2/5. Synovial biopsy suggested septic arthritis in 5 out of 6. These cases of pseudoseptic arthritis were indistinguishable from true septic arthritis. Follow-up is required in septic arthritis with negative culture findings to exclude rheumatic disease.

Septic arthritis with negative bacteriological findings in adult native joints: a retrospective study of 74 cases.

BACKGROUND: No microorganism is identified in 7-35% of cases of septic arthritis. The diagnosis is, therefore, only presumptive. We reviewed our cases of septic arthritis in adult native joints to determine the frequency of negative cultures, disease characteristics and the frequency of misdiagnosis of septic arthritis. METHODS: This retrospective study included all patients admitted to our department from 1979-2005 with arthritis, diagnosed and treated as septic. RESULTS: No microorganism was isolated from synovial fluid or blood samples from 74 out of 398 (19%) patients with presumed septic arthritis. Patients without microorganisms were younger (54 vs 62 years), less likely to have risk factors for septic arthritis (31% vs 41%) and had lower mortality (0 vs 5%) than patients with positive cultures. Long-term outcome was known for 48 patients. A retrospective analysis of all data and long-term outcome concluded that septic arthritis was probable in 18 patients and improbable in 13. Ten of the latter developed rheumatic disease after a mean time of 6 months: rheumatoid arthritis (n=3), spondyloarthropathies (n=3), unclassified rheumatic disease (n=2), Wegener granulomatosis (n=1) and cytosteatonecrosis (n=1). Fever and signs of inflammation were more frequent and synovial fluid cell counts were higher in patients with improbable septic arthritis. Conversely, radiological signs were more common in patients with probable septic arthritis. CONCLUSION: At least 14% of patients diagnosed with septic arthritis with negative bacteriological results subsequently develop rheumatic disease. This pseudoseptic arthritis is indistinguishable from true septic arthritis. When no microorganism is identified, the diagnosis remains presumptive and follow-up is necessary to screen for other diseases, especially rheumatic diseases.