Blau syndrome (BS) is an auto-inflammatory granulomatous disease that possibly involves abnormal response to interferon gamma (IFNγ) due to exaggerated nucleotide-binding oligomerization domain containing 2 (NOD2) activity. Mendelian susceptibility to mycobacterial diseases (MSMD) is an infectious granulomatous disease that is caused by impaired production of or response to IFNγ. We report a mother and daughter who are both heterozygous for NOD2c.2264C˃T variant and dominant-negative IFNGR1818del4 mutation. The 17-year-old patient displayed an altered form of BS and milder form of MSMD, whereas the 44-year-old mother was completely asymptomatic. This experiment of nature supports the notion that IFNγ is an important driver of at least some BS manifestations and that elucidation of its involvement in the disease immunopathogenesis may identify novel therapeutic targets.

• Keywords
• Blau syndrome, IFNγ, IFNγR1, MSMD, NOD2, WES, methotrexate,
• MeSH
• Arthritis genetics   MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Mutation genetics   MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon deficiency   genetics   MeSH
• Sarcoidosis genetics   MeSH
• Nod2 Signaling Adaptor Protein genetics   MeSH
• Synovitis genetics   MeSH
• Uveitis genetics   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NOD2 protein, human MeSH Browser
• Receptors, Interferon MeSH
• Nod2 Signaling Adaptor Protein MeSH

Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.

• MeSH
• Interferon-gamma chemistry   metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Models, Molecular * MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon chemistry   genetics   metabolism   MeSH
• Protein Folding * MeSH
• Amino Acid Substitution * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IFNG protein, human MeSH Browser
• Interferon-gamma MeSH
• Receptors, Interferon MeSH

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.

• MeSH
• Interferon-gamma chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Surface Plasmon Resonance MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon chemistry   genetics   metabolism   MeSH
• Protein Folding * MeSH
• Molecular Dynamics Simulation * MeSH
• Amino Acid Substitution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IFNG protein, human MeSH Browser
• Interferon-gamma MeSH
• Receptors, Interferon MeSH

AIMS: To follow the IFNγ receptor expression on monocytes and granulocytes of cardiac surgical patients with respect to the type of cardiopulmonary bypass (CPB). METHODS: Expression of IFNγ receptor on monocytes and granulocytes of 26 cardiac surgical patients operated with the use of either "standard" or "miniaturised" CPB was determined by flow cytometry. RESULTS: The significant increase in IFNγ receptor expression on monocytes on the 1(st) and on the 3(rd) postoperative days was revealed in both groups of patients (p<0.001) irrespective of the type of CPB used, being non-significantly different between groups. In contrast, the expression of IFNγ on granulocytes displayed significant differences in terms of the CPB used. Whereas, in "standard" CPB patients, granulocyte INFγ receptor expression reached its maximum immediately after surgery (p<0.01), in "miniivasive" CPB patients, the peak in INFγ receptor expression was postponed to the 1(st) postoperative day (p<0.05). Statistically significantly higher IFNγ receptor expression on granulocytes was found in "standard" CPB patients (p<0.05). CONCLUSION: Compared to "miniaturised" CPB patients, the significantly higher IFNγ receptor expression on granulocytes was found in "standard" CPB patients (p<0.05) on the 1(st) postoperative day.

• MeSH
• Granulocytes metabolism   MeSH
• Cardiac Surgical Procedures * MeSH
• Cardiopulmonary Bypass classification   methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Miniaturization MeSH
• Monocytes metabolism   MeSH
• Postoperative Period MeSH
• Flow Cytometry MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon metabolism   MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Receptors, Interferon MeSH

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.

• MeSH
• Cell Line MeSH
• Chlorocebus aethiops MeSH
• Gene Deletion * MeSH
• DNA Primers MeSH
• Interferon-gamma physiology   MeSH
• Rabbits MeSH
• Humans MeSH
• Mice MeSH
• Polymerase Chain Reaction MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon deficiency   genetics   physiology   MeSH
• Vaccinia immunology   MeSH
• Virulence MeSH
• Vaccinia virus pathogenicity   physiology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• Interferon-gamma MeSH
• Receptors, Interferon MeSH