Factors affecting protein recovery during Hsp40 affinity profiling.

The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.

Applications of red light photoredox catalysis in organic synthesis.

Photoredox catalysis has emerged as an efficient and versatile approach for developing novel synthetic methodologies. Particularly, red light photocatalysis has attracted more attention due to its intrinsic advantages of low energy, few health risks, few side reactions, and high penetration depth through various media. Impressive progress has been made in this field. In this review, we outline the applications of different photoredox catalysts in a wide range of red light-mediated reactions including direct red light photoredox catalysis, red light photoredox catalysis through upconversion, and dual red light photoredox catalysis. Due to the similarities between near-infrared (NIR) and red light, an overview of NIR-induced reactions is also presented. Lastly, current evidence showing the advantages of red light and NIR photoredox catalysis is also described.

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling.

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting and, in turn, threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), is difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for both multiplexed protein labeling and for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we discover that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure.

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that a brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis and to identify the susceptible proteome.

Synthesis of CF3-Containing Spirocyclic Indolines via a Red-Light-Mediated Trifluoromethylation/Dearomatization Cascade.

A red-light-mediated nPr-DMQA+-catalyzed cascade intramolecular trifluoromethylation and dearomatization of indole derivatives with Umemoto's reagent has been developed. This protocol provides a facile and efficient approach for the construction of functionalized and potentially biologically important CF3-containing 3,3-spirocyclic indolines with moderate to high yields and excellent diastereoselectivities under mild conditions. The success of multiple gram-scale (1 and 10 g) experiments further highlights the robustness and practicality of this protocol and the merit of the employment of red light. Mechanistic studies support the formation of a crucial CF3 radical species and a dearomatized benzyl carbocation intermediate.

Helical Carbenium Ion: A Versatile Organic Photoredox Catalyst for Red-Light-Mediated Reactions.

Red light has the advantages of low energy, less health risks, and high penetration depth through various media. Herein, a helical carbenium ion (N,N'-di-n-propyl-1,13-dimethoxyquinacridinium (nPr-DMQA+) tetrafluoroborate) has been used as an organic photoredox catalyst for photoreductions and photooxidations in the presence of red light (λmax = 640 nm). It has catalyzed red-light-mediated dual transition-metal/photo-redox-catalyzed C-H arylation and intermolecular atom-transfer radical addition through oxidative quenching. Moreover, its potential in photooxidation catalysis has also been demonstrated by successful applications in red-light-induced aerobic oxidative hydroxylation of arylboronic acids and benzylic C(sp3)-H oxygenation through reductive quenching. Thus, a versatile organic photoredox catalyst (helical carbenium ion) for red-light-mediated photoredox reactions has been developed.

Tunable carbocation-based redox active ambiphilic ligands: synthesis, coordination and characterization.

The synthesis of novel redox active ambiphilic ligands L1-L3 and their coordination chemistry to first-row late transition metal halides (M = Co and Ni) is reported. The heterocyclic carbocation scaffolds act as Lewis acid moieties while the pyridine anchor acts as the coordinating Lewis base. The high synthetic tunability of this ligand scaffold allows for control of its rigidity and electronic properties. Anion exchange and coordination of a chloride anion to the metal center was observed resulting in the formation of [MCl3]- metallate. Upon coordination to the pyridine anchor, the metallate centers adopt a canonical tetrahedral geometry, resulting in an overall neutral complex best described as a zwitterionic metallate trichloride bound to a cationic ligand. Characterization techniques including single crystal X-ray diffraction, cyclic voltammetry, and UV-Vis absorption spectroscopy were employed to better understand the structural and chemical properties of the ligands and metal complexes. A possible weak interaction between one of the chlorides and the carbenium moiety in the ligand is observed in crystals of both of the Co(ii) and Ni(ii) complexes with ligand L1. Density functional theory (DFT) calculations support that this electrostatic interaction for complexes 2a and 2b exists only in the solid state.

Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry.

The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

Molecular Orbital Insights of Transition Metal-Stabilized Carbocations.

Transition metal-stabilized carbocations are characterized by synthetically valuable interactions, yet, to date there are no comprehensive reports of the many bonding modes that can exist between a metal and carbocation. This review summarizes developments in these complexes to provide a clear picture of their properties and reactivities. In order to strategically exploit them, we propose this summary of the different bonding modes for transition metal-carbocation complexes. These models will help chemists understand the orbital interactions involved in these compounds so that they can approach their synthetic goals most effectively. Multiple transition metals and carbocations will be discussed.

Catalyst-Dependent Stereodivergent and Regioselective Synthesis of Indole-Fused Heterocycles through Formal Cycloadditions of Indolyl-Allenes.

Stereo- and regioselective construction of poly-heterocycles, especially those with several contiguous stereocenters, is still a challenge. In this paper, catalyst-dependent stereodivergent and regioselective synthesis of indole-fused heterocycles through formal cycloadditions of indolyl-allenes has been developed. The reaction features total reversion of an all-carbon quaternary stereocenter when a gold or platinum complex was employed as the catalyst through [3 + 2] cycloaddition of allene with indole, affording different diazabenzo[a]cyclopenta[cd]azulenes as epimers, respectively. In addition, in the presence of IPrAuCl and AgNTf2, highly regioselective exo-type [2 + 2] cycloaddition was observed, in which allene served as a 2C synthon. This methodology provides a simple and straightforward approach for the construction of indole-fused tricyclic systems under mild conditions in an atom-economical way.

One-pot tandem diastereoselective and enantioselective synthesis of functionalized oxindole-fused spiropyrazolidine frameworks.

A highly efficient palladium(0)-catalyzed asymmetric [3+2] cycloaddition using 3-diazooxindoles serving as dipolarophiles affords functionalized pyrazolidine derivatives in an atom-economical way. In addition, by trapping the pyrazolidine derivatives with maleimides, the corresponding spiropyrazolidine oxindoles containing multiple stereogenic centers have been obtained in high yields along with moderate to good levels of diastereoselectivity and enantioselectivity under mild conditions. Thus, a novel three-component one-pot tandem reaction has been developed.

Rhodium-catalyzed three-component reaction of 3-diazooxindoles with indoles and isatin-derived ketimines: a facile and versatile approach to functionalized 3,3',3''-trisindoles.

A simple, facile and efficient Rh2(OAc)4-catalyzed three-component reaction of 3-diazooxindoles with indoles and isatin-derived N-Boc ketimines towards a variety of functionalized 3,3',3''-trisindoles in high yields with moderate to excellent diastereoselectivities has been developed. This methodology provides an ideal approach for the direct introduction of indole and oxindole into an isatin moiety at the 3-position.

Asymmetric catalytic Mannich-type reaction of hydrazones with difluoroenoxysilanes using imidazoline-anchored phosphine ligand-zinc(II) complexes.

Asymmetric Mannich-type reaction of hydrazones with difluoroenoxysilanes using chiral zinc(II)-imidazoline-phosphine complexes as catalysts have been established, giving the corresponding adducts in good to excellent enantioselectivity and chemical yields under mild conditions.