Notes

Cadaveric Review regarding Crista Fenestra: Revisited.
The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). Vorinostat The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is wbed using other labeling methods that stabilize these interactions.Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.Effective delivery of proteins into the cytosol of mammalian cells would open the door to a wide range of applications. However, despite great efforts from numerous investigators, effective protein delivery in a clinical setting is yet to be accomplished. Herein we report a potentially general approach to engineering cell-permeable proteins by genetically grafting a short cell-penetrating peptide (CPP) to an exposed loop of a protein of interest. The grafted peptide is conformationally constrained, exhibiting enhanced proteolytic stability and cellular entry efficiency. Applying this technique to enhanced green fluorescent protein (EGFP), protein-tyrosine phosphatase 1B (PTP1B), and purine nucleoside phosphorylase (PNP) rendered all three proteins cell-permeable and biologically active in cellular assays. When added into growth medium at 0.5-5 μM concentrations, the engineered PTP1B dose-dependently reduced the phosphotyrosine levels of intracellular proteins, while the modified PNP corrected the metabolic deficiency of PNP-deficient mouse T lymphocytes, providing a potential enzyme replacement therapy for a rare genetic disease.Significant advancement of chemoproteomics has contributed to uncovering the mechanism of action (MoA) of small-molecule drugs by characterizing drug-protein interactions in living systems. However, cell-membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels, due to their low abundance and unique biophysical properties associated with multiple transmembrane domains, can present challenges for proteome-wide mapping of drug-receptor interactions. Herein, we describe the development of novel tetrafunctional probes, consisting of (1) a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive group, (3) a hydrazine-labile cleavable linker, and (4) biotin for enrichment. In live cell labeling studies, we demonstrated that the ACT-based probe showed superior reactivity and selectivity for labeling on-target GPCR by mass spectrometry analysis compared with control probes including diazirine-based probes. By leveraging ACT-based cleavable probes, we further identified a set of representative ionotropic receptors, targeted by CNS drugs, with remarkable selectivity and precise binding site information from mouse brain slices. We anticipate that the robust chemoproteomic platform using the ACT-based cleavable probe coupled with phenotypic screening should promote identification of pharmacologically relevant target receptors of drug candidates and ultimately development of first-in-class drugs with novel MoA.The high temperature requirement A (HTRA) family of serine proteases mediates protein quality control. These proteins process misfolded proteins in several diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). While their structures and activation mechanisms have been studied, the precise details of the regulation of their activity under physiological conditions have not been completely elucidated, partly due to the lack of suitable chemical probes. In the present study, we developed novel activity-based probes (ABPs) targeting the HTRAs and demonstrated their utility in the monitoring and quantification of changes in enzyme activity in live cells. Using our probes, we found the activity of HTRA1 to be highly elevated in an AD-like cell-based model. We also observed the active HTRA2 in live cells by using a mitochondrion-targeted probe. We believe that our probes can serve as a useful tool to study the role of human HTRAs in neurodegenerative diseases.
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