Project description:14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development. Biophysical methodologies are an essential element of the 14-3-3 analytical toolbox, but in many cases have not been fully exploited. Here, we present a contemporary review of the predominant biophysical techniques used to study 14-3-3 protein-protein interactions, with a focus on examples that address key questions and challenges in the 14-3-3 field.
Project description:Human biology is regulated by a complex network of protein-protein interactions (PPIs), and disruption of this network has been implicated in many diseases. However, the targeting of PPIs remains a challenging area for chemical probe and drug discovery. Although many methodologies have been put forth to facilitate these efforts, new technologies are still needed. Current biochemical assays for PPIs are typically limited to motif-domain and domain-domain interactions, and assays that will enable the screening of full-length protein systems, which are more biologically relevant, are sparse. To overcome this barrier, we have developed a new assay technology, "PPI catalytic enzyme-linked click chemistry assay" or PPI cat-ELCCA, which utilizes click chemistry to afford catalytic signal amplification. To validate this approach, we have applied PPI cat-ELCCA to the eIF4E-4E-BP1 and eIF4E-eIF4G PPIs, key regulators of cap-dependent mRNA translation. Using these examples, we have demonstrated that PPI cat-ELCCA is amenable to full-length proteins, large (>200 kDa) and small (∼12 kDa), and is readily adaptable to automated high-throughput screening. Thus, PPI cat-ELCCA represents a powerful new tool in the toolbox of assays available to scientists interested in the targeting of disease-relevant PPIs.
Project description:In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors.
Project description:Latest estimates indicate that nutritional deficiencies account for 3 million child deaths each year in less-developed countries. Targeted nutritional interventions could therefore save millions of lives. However, such interventions require careful optimization to maximize benefit and avoid harm. Progress toward designing effective life-saving interventions is currently hampered by some serious gaps in our understanding of nutrient metabolism in humans. In this Personal Perspective, we highlight some of these gaps and make some proposals as to how improved research methods and technologies can be brought to bear on the problems of undernourished children in the developing world.
Project description:The micro-exon genes (MEG) of Schistosoma mansoni, a parasite responsible for the second most widely spread tropical disease, code for small secreted proteins with sequences unique to the Schistosoma genera. Bioinformatics analyses suggest the soluble domain of the MEG-14 protein will be largely disordered, and using synchrotron radiation circular dichroism spectroscopy, its secondary structure was shown to be essentially completely unfolded in aqueous solution. It does, however, show a strong propensity to fold into more ordered structures under a wide range of conditions. Partial folding was produced by increasing temperature (in a reversible process), contrary to the behavior of most soluble proteins. Furthermore, significant folding was observed in the presence of negatively charged lipids and detergents, but not in zwitterionic or neutral lipids or detergents. Absorption onto a surface followed by dehydration stimulated it to fold into a helical structure, as it did when the aqueous solution was replaced by nonaqueous solvents. Hydration of the dehydrated folded protein was accompanied by complete unfolding. These results support the identification of MEG-14 as a classic intrinsically disordered protein, and open the possibility of its interaction/folding with different partners and factors being related to multifunctional roles and states within the host.
Project description:The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrP(C)), to proteolytic digestion has been well documented. In addition, a link between PrP(C) and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrP(C)-expressing mice were treated with calpain, no appreciable loss of the intact PrP(C), nor the appearance of PrP(C) breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrP(C)-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrP(C) is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrP(C). Similarly, the lysosomal protease inhibitor, NH(4)Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrP(C) or its release from the RtCNC. In conclusion, rat neuronal PrP(C) is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrP(C) into the cell culture media suggests an unidentified neuroprotective mechanism for PrP(C).
Project description:We have developed a novel assay system for systematic analysis of protein-protein interactions (PPIs) that is characteristic of a PCR-mediated rapid sample preparation and a high-throughput assay system based on the mammalian two-hybrid method. Using gene-specific primers, we successfully constructed the assay samples by two rounds of PCR with up to 3.6 kb from the first-round PCR fragments. In the assay system, we designed all the steps to be performed by adding only samples, reagents, and cells into 384-well assay plates using two types of semiautomatic multiple dispensers. The system enabled us examine more than 20,000 assay wells per day. We detected 145 interactions in our pilot study using 3500 samples derived from mouse full-length enriched cDNAs. Analysis of the interaction data showed both several significant interaction clusters and predicted functions of a few uncharacterized proteins. In combination with our comprehensive mouse full-length cDNA clone bank covering a large part of the whole genes, our high-throughput assay system will discover many interactions to facilitate understanding of the function of uncharacterized proteins and the molecular mechanism of crucial biological processes, and also enable completion of a rough draft of the entire PPI panel in certain cell types or tissues of mouse within a short time.
Project description:Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as "undruggable" targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products.
Project description:Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.
Project description:This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.