Dynamic Structural Differences between Human and Mouse STING Lead to Differing Sensitivity to DMXAA

The stimulator-of-interferon-genes (STING) protein is involved in innate immunity. It has recently been shown that modulation of STING can lead to an aggressive antitumor response. DMXAA is an antitumor agent that had shown great promise in murine models but failed in human clinical trials. The molecular target of DMXAA was subsequently shown to be murine STING (mSTING); however, human STING (hSTING) is insensitive to DMXAA. Molecular dynamics simulations were employed to investigate the differences between hSTING and mSTING that could influence DMXAA binding. An initial set of simulations was performed to investigate a single lid region mutation G230I in hSTING (corresponding residue in mSTING is an Ile), which rendered the protein sensitive to DMXAA. The simulations found that an Ile side chain was enough to form a steric barrier that prevents exit of DMXAA, whereas in WT hSTING, the Gly residue that lacks a side chain formed a porous lid region that allowed DMXAA to exit. A second set of molecular dynamics simulations compared the tendency of STING to be in an open-inactive conformation or a closed-active conformation. The results show that hSTING prefers to be in an open-inactive conformation even with cGAMP, the native ligand, bound. On the other hand, mSTING prefers a closed-active conformation even without a ligand bound. These results highlight the challenges in translating a mouse active STING compound into a human active compound, while also providing avenues to pursue for designing a small-molecule drug targeting human STING.     

Destabilizing the AXH Tetramer by Mutations: Mechanisms and Potential Antiaggregation Strategies

The AXH domain of protein Ataxin 1 is thought to play a key role in the misfolding and aggregation pathway responsible for Spinocerebellar ataxia 1. For this reason, a molecular level understanding of AXH oligomerization pathway is crucial to elucidate the aggregation mechanism, which is thought to trigger the disease. This study employs classical and enhanced molecular dynamics to identify the structural and energetic basis of AXH tetramer stability. Results of this work elucidate molecular mechanisms behind the destabilizing effect of protein mutations, which consequently affect the AXH tetramer assembly. Moreover, results of the study draw attention for the first time, to our knowledge, to the R638 protein residue, which is shown to play a key role in AXH tetramer stability. Therefore, R638 might be also implicated in the AXH oligomerization pathway and stands out as a target for future experimental studies focused on self-association mechanisms and fibril formation of full-length ATX1.     

Instability of microsatellites linked to targeted genes in CRISPR/Cas9-edited and traditional gene knockout mouse strains

正The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,although it has been popular during the past decades.Recent years,genome editing which can cause DNA sequence-specific mutations in the genomes of cellular     

Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1’s lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.     

Mutations in CSPP1 Lead to Classical Joubert Syndrome

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.