CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

1. Download : Download high-res image (183KB)
2. Download : Download full-size image

     

Discovery of dual ZAP70 and Syk kinases inhibitors by docking into a rare C-helix-out conformation of Syk

The non-receptor tyrosine kinase Syk (spleen tyrosine kinase) is a pharmaceutical relevant target because its over-activation is observed in several autoimmune diseases, allergy, and asthma. Here we report the identification of two novel inhibitors of Syk by high-throughput docking into a rare C-helix-out conformation published recently. Interestingly, both compounds are slightly more active on ZAP70 (Zeta-chain-associated protein kinase 70), which is the kinase closest to Syk in the phylogenetic tree of human kinases. Taken together, the docking pose and experimental results suggest that the higher affinity of the inhibitors for ZAP70 than Syk originates from a more populated C-helix-out conformation in ZAP70. The latter observation is congruent with the 100-fold lower intrinsic activity of ZAP70 than Syk, as the C-helix-out conformation is inactive. The pharmacophore features of DFG-in, C-helix-out compounds are analyzed in relation to DFG-out inhibitors.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Homodimerization as a molecular switch between low and high efficiency PrPC cell surface delivery and neuroprotective activity

PrP^C is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP^C trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP^C-derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aβ molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP^C trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP^C homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.     

A simple,band‐mounted device to measure behaviorally modified thermal microclimates experienced by birds

Birds encounter climate at the scale of microclimates that can vary rapidly in time and space and so understanding potential vulnerability and adaptations to those microclimates requires fine‐scale measurements that accurately track thermal exposures. However, few options exist for recording the microclimates actually experienced by birds (realized microclimates). We constructed and tested a simple, low‐cost, temperature logger for recording the realized microclimates of ground‐nesting birds. We developed attachment protocols for band‐mounting Thermochron iButtons on Ring‐billed Gull (Larus delawarensis) chicks. We tested these mounted, temperature‐logging devices on 20 chicks weighing > 200 g (device weight was 4 g), attaching devices for 48 h and observing behavior before and after attachment and removal. Devices recorded temperature immediately surrounding the leg at 2‐min intervals. Recorded temperatures were strong predictors of observed thermoregulatory behaviors (panting and sitting), outperforming predictions based on air temperatures measured by basic, static approaches. Through comparison with matched controls (chicks with just a band), we detected no adverse physiological effects of devices, no effects on social or feeding behavior, and only a short‐term decrease in inactivity immediately after device attachment (likely due to increased preening). By attaching iButtons to the legs of birds, we quantified realized thermal exposure, integrating air temperature, modes of environmental heat transfer, and bird behavior at microclimatic scales. Although not yet validated for broader use, our approach (including possible miniaturization) should be suitable to measure thermal exposure of adults, not just chicks, allowing collection of data concerning thermal exposures during flight under field conditions. At ~ $25 USD per device, our approach facilitates experimental protocols with robust sample sizes, even for relatively modest budgets.     

Functional assignment to JEV proteins using SVM

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).     

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.     

Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.