The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin

Viperin, an antiviral protein, has been shown to contain a CX3CX2C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α -β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity

Immune checkpoint inhibitor PD-1 pathway is down-regulated in synovium at various stages of rheumatoid arthritis disease progression.

Immune checkpoint blockade with therapeutic anti-cytotoxic T lymphocyte-associated antigen (CTLA)-4 (Ipilimumab) and anti-programmed death (PD)-1 (Nivolumab and Pembrolizumab) antibodies alone or in combination has shown remarkable efficacy in multiple cancer types, concomitant with immune-related adverse events, including arthralgia and inflammatory arthritis (IA) in some patients. Herein, using Nivolumab (anti-PD-1 antagonist)-responsive genes along with transcriptomics of synovial tissue from multiple stages of rheumatoid arthritis (RA) disease progression, we have interrogated the activity status of PD-1 pathway during RA development. We demonstrate that the expression of PD-1 was increased in early and established RA synovial tissue compared to normal and OA synovium, whereas that of its ligands, programmed death ligand-1 (PD-L1) and PD-L2, was increased at all the stages of RA disease progression, namely arthralgia, IA/undifferentiated arthritis, early RA and established RA. Further, we show that RA patients expressed PD-1 on a majority of synovial tissue infiltrating CD4+ and CD8+ T cells. Moreover, enrichment of Nivolumab gene signature was observed in IA and RA, indicating that the PD-1 pathway was downregulated during RA disease progression. Furthermore, serum soluble (s) PD-1 levels were increased in autoantibody positive early RA patients. Interestingly, most of the early RA synovium tissue sections showed negative PD-L1 staining by immunohistochemistry. Therefore, downregulation in PD-1 inhibitory signaling in RA could be attributed to increased serum sPD-1 and decreased synovial tissue PD-L1 levels. Taken together, these data suggest that agonistic PD1 antibody-based therapeutics may show efficacy in RA treatment and interception

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab

Multilevel genomics of colorectal cancers with microsatellite instability—clinical impact of JAK1 mutations and consensus molecular subtype 1

Background Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations—the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. Methods A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling—including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. Results Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transcriptional signatures associated with resistance to anti-PD-1 treatment. Clonality analyses revealed a high level of intra-tumor heterogeneity; however, this was not associated with disease progression. Among the MSI+ tumors, the total mutation load correlated with the number of predicted neoantigens (P = 4 × 10−5), but not with immune cell infiltration—this was dependent on the CMS class; MSI+ tumors in CMS1 were highly immunogenic compared to MSI+ tumors in CMS2-4. Both JAK1 mutations and CMS1 were favorable prognostic factors (hazard ratios 0.2 [0.05–0.9] and 0.4 [0.2–0.9], respectively, P = 0.03 and 0.02). Conclusions Multilevel genomic analyses of MSI+ colorectal cancer revealed molecular heterogeneity with clinical relevance, including tumor immunogenicity and a favorable patient outcome associated with JAK1 mutations and the transcriptomic subgroup CMS1, emphasizing the potential for prognostic stratification of this clinically important subtype. See related research highlight by Samstein and Chan 10.1186/s13073-017-0438-

Binding of 5′-GTP to the C-terminal FeS cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism

The first step in molybdenum cofactor biosynthesis, the conversion of 5′-GTP to precursor Z, an oxygen-sensitive tetrahydropyranopterin is catalyzed by the S-adenosylmethionine (SAM)-dependent enzyme MoaA and the accessory protein MoaC. This reaction involves the radical-initiated intramolecular rearrangement of the guanine C8 atom. MoaA harbors an N-terminal [4Fe–4S] cluster, which is involved in the reductive cleavage of SAM and generates a 5′-deoxyadenosyl radical (5′-dA(•)), and a C-terminal [4Fe–4S] cluster presumably involved in substrate binding and/or activation. Biochemical studies identified residues involved in 5′-GTP binding and the determinants of nucleotide specificity. The crystal structure of MoaA in complex with 5′-GTP confirms the biochemical data and provides valuable insights into the subsequent radical reaction. MoaA binds 5′-GTP with high affinity and interacts through its C-terminal [4Fe–4S] cluster with the guanine N1 and N2 atoms, in a yet uncharacterized binding mode. The tightly anchored triphosphate moiety prevents the escape of radical intermediates. This structure also visualizes the l-Met and 5′-dA cleavage products of SAM. Rotation of the 5′-dA ribose and/or conformational changes of the guanosine are proposed to bring the 5′-deoxyadenosyl radical into close proximity of either the ribose C2′ and C3′ or the guanine C8 carbon atoms leading to hydrogen abstraction

Crystal structure of the S-adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans

The MoaA and MoaC proteins catalyze the first step during molybdenum cofactor biosynthesis, the conversion of a guanosine derivative to precursor Z. MoaA belongs to the S-adenosylmethionine (SAM)-dependent radical enzyme superfamily, members of which catalyze the formation of protein and/or substrate radicals by reductive cleavage of SAM by a [4Fe–4S] cluster. A defined in vitro system is described, which generates precursor Z and led to the identification of 5′-GTP as the substrate. The structures of MoaA in the apo-state (2.8 Å) and in complex with SAM (2.2 Å) provide valuable insights into its mechanism and help to define the defects caused by mutations in the human ortholog of MoaA that lead to molybdenum cofactor deficiency, a usually fatal disease accompanied by severe neurological symptoms. The central core of each subunit of the MoaA dimer is an incomplete triosephosphate isomerase barrel formed by the N-terminal part of the protein, which contains the [4Fe–4S] cluster typical for SAM-dependent radical enzymes. SAM is the fourth ligand to the cluster and binds to its unique Fe as an N/O chelate. The lateral opening of the incomplete triosephosphate isomerase barrel is covered by the C-terminal part of the protein containing an additional [4Fe–4S] cluster, which is unique to MoaA proteins. Both FeS clusters are separated by ≈17 Å, with a large active site pocket between. The noncysteinyl-ligated unique Fe site of the C-terminal [4Fe–4S] cluster is proposed to be involved in the binding and activation of 5′-GTP