Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition

AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly

Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu).Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work.Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.Peer Reviewe

Harvesting Candidate Genes Responsible for Serious Adverse Drug Reactions from a Chemical-Protein Interactome

Identifying genetic factors responsible for serious adverse drug reaction (SADR) is of critical importance to personalized medicine. However, genome-wide association studies are hampered due to the lack of case-control samples, and the selection of candidate genes is limited by the lack of understanding of the underlying mechanisms of SADRs. We hypothesize that drugs causing the same type of SADR might share a common mechanism by targeting unexpectedly the same SADR-mediating protein. Hence we propose an approach of identifying the common SADR-targets through constructing and mining an in silico chemical-protein interactome (CPI), a matrix of binding strengths among 162 drug molecules known to cause at least one type of SADR and 845 proteins. Drugs sharing the same SADR outcome were also found to possess similarities in their CPI profiles towards this 845 protein set. This methodology identified the candidate gene of sulfonamide-induced toxic epidermal necrolysis (TEN): all nine sulfonamides that cause TEN were found to bind strongly to MHC I (Cw*4), whereas none of the 17 control drugs that do not cause TEN were found to bind to it. Through an insight into the CPI, we found the Y116S substitution of MHC I (B*5703) enhances the unexpected binding of abacavir to its antigen presentation groove, which explains why B*5701, not B*5703, is the risk allele of abacavir-induced hypersensitivity. In conclusion, SADR targets and the patient-specific off-targets could be identified through a systematic investigation of the CPI, generating important hypotheses for prospective experimental validation of the candidate genes

Exploring Off-Targets and Off-Systems for Adverse Drug Reactions via Chemical-Protein Interactome — Clozapine-Induced Agranulocytosis as a Case Study

In the era of personalized medical practice, understanding the genetic basis of patient-specific adverse drug reaction (ADR) is a major challenge. Clozapine provides effective treatments for schizophrenia but its usage is limited because of life-threatening agranulocytosis. A recent high impact study showed the necessity of moving clozapine to a first line drug, thus identifying the biomarkers for drug-induced agranulocytosis has become important. Here we report a methodology termed as antithesis chemical-protein interactome (CPI), which utilizes the docking method to mimic the differences in the drug-protein interactions across a panel of human proteins. Using this method, we identified HSPA1A, a known susceptibility gene for CIA, to be the off-target of clozapine. Furthermore, the mRNA expression of HSPA1A-related genes (off-target associated systems) was also found to be differentially expressed in clozapine treated leukemia cell line. Apart from identifying the CIA causal genes we identified several novel candidate genes which could be responsible for agranulocytosis. Proteins related to reactive oxygen clearance system, such as oxidoreductases and glutathione metabolite enzymes, were significantly enriched in the antithesis CPI. This methodology conducted a multi-dimensional analysis of drugs' perturbation to the biological system, investigating both the off-targets and the associated off-systems to explore the molecular basis of an adverse event or the new uses for old drugs

American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18