Carlos A. Gartner

Enhancement of proteasome activity by a small-molecule inhibitor of USP14

Proteasomes, the primary mediators of ubiquitin–protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin–protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Proteomic Analysis of GLUT4 Storage Vesicles Reveals LRP1 to Be an Important Vesicle Component and Target of Insulin Signaling

Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[3H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.

Photosensitizers: comprehensive photophysics/photochemistry and theory of coumarins, chromones, their homologues and thione analogues

The photophysical–photochemical behaviour, including absorption spectra, fluorescence spectra and lifetimes, phosphorescence spectra, triplet–triplet transient spectra, triplet lifetimes and quantum yields and sensitized singlet oxygen formation and yields, has been determined for many of a group of 22 carbonyl and thione compounds. These include coumarins, psoralens, chromones and furochromone and their thiones. Many of the thiones were synthesized for the first time. Two types of theoretical calculations were carried out on the majority of the compounds. The principal goals of the research for the thiones, were (1) to produce or improve the photosensitizer capabilty of the compounds in vivo, (2) to shift the spectra of the compounds to significantly longer wavelength and (3) to increase the triplet and singlet oxygen quantum yields. Most of these have been largely achieved. The photosensitizing abilities of two of the thiones have been evaluated using a unique testing technique employing a series of genetically engineered bacteria.

Assessing Enzyme Activities Using Stable Isotope Labeling and Mass Spectrometry

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.

Identification of FBXO25-interacting proteins using an integrated proteomics approach

FBXO25 is one of the 68 human F‐box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s‐phase‐kinase associated protein 1, really interesting new gene‐box 1, Cullin 1, and F‐box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25‐associated nuclear domains (FAND). Combining two‐step affinity purification followed by MS with a classical two‐hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, β‐actin, physically interacts through its N‐terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.