A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

Muscle atrophy prevails in numerous diseases (cancer cachexia, renal failure, infections, etc. ), mainly results from elevated proteolysis, and is accelerated by bed rest. This largely contributes to increased health costs. Devising new strategies to prevent muscle wasting is a major clinical challenge. The ubiquitin proteasome system (UPS) degrades myofibrillar proteins, but the precise mechanisms responsible for actin breakdown are surprisingly poorly characterized. We report that chimeric flag‐actin was destabilized and polyubiquitinylated in stably transfected C2C12 myotubes treated with the catabolic agent dexa‐methasone (1 μM) and that only proteasome inhibitors blocked its breakdown. Actin polyubiquitinylation was also detected in wild‐type C2C12 myotubes and human muscle biopsies from control participants and patients with cancer. The muscle‐specific E3 ubiquitin ligase MuRF1 is up‐regulated in catabolic conditions and polyubiquitinylates components of the thick filament. We also demonstrate that recombinant GST‐MuRF1 physically interacted and polyubiquitinylated actin in vitro and that MuRF1 is a critical component for actin breakdown, since MuRF1 siRNA stabilized flag‐actin. These data identify unambiguously the abundant contractile protein actin as a target of the UPS in skeletal muscle both in vitro and in vivo , further supporting the need for new strategies blocking specifically the activation of this pathway in muscle wasting conditions.—Polge, C., Heng, A.‐E., Jarzaguet, M., Ventadour, S., Claustre, A., Combaret, L., Béchet, D., Matondo, M., Uttenweiler‐Joseph, S., Monsarrat, B., Attaix, D., Taillandier, D. Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1. FASEB J. 25, 3790–3802 (2011). www.fasebj.org

The E3 ubiquitin ligase MuRF1/TRIM63 was identified 20 years ago and suspected to play important roles during skeletal muscle atrophy. Since then, numerous studies have been conducted to decipher the roles, molecular mechanisms and regulation of this enzyme. This revealed that MuRF1 is an important player in the skeletal muscle atrophy process occurring during catabolic states, making MuRF1 a prime candidate for pharmacological treatments against muscle wasting. Indeed, muscle wasting is an associated event of several diseases (e.g., cancer, sepsis, diabetes, renal failure, etc.) and negatively impacts the prognosis of patients, which has stimulated the search for MuRF1 inhibitory molecules. However, studies on MuRF1 cardiac functions revealed that MuRF1 is also cardioprotective, revealing a yin and yang role of MuRF1, being detrimental in skeletal muscle and beneficial in the heart. This review discusses data obtained on MuRF1, both in skeletal and cardiac muscles, over the past 20 years, regarding the structure, the regulation, the location and the different functions identified, and the first inhibitors reported, and aim to draw the picture of what is known about MuRF1. The review also discusses important MuRF1 characteristics to consider for the design of future drugs to maintain skeletal muscle mass in patients with different pathologies.

One of the most noticeable effects of aging is the reduction in skeletal muscle mass and strength (sarcopenia). The metabolic syndrome (MS) is also prevalent in old subjects, but its relevance to skeletal muscle characteristics has poorly been investigated. Immunohistochemical studies were performed with muscle biopsies from young (22 years) and old (73 years) men with and without MS to reveal age-dependent and MS-associated modifications of fiber-type characteristics. Atrophy of type II fibers and altered fiber shape characterized muscle aging in lean healthy men. In contrast, increased cross-sectional area of the most abundant type I and type IIA fibers, and reduced cytochrome c oxidase content in all fiber types, characterized MS. Aging and particularly MS were associated with accumulation of intramyocellular lipid droplets. Although lipids mostly accumulated in type I fibers, matrix-assisted laser desorption/ionization–mass spectrometry imaging of intramyocellular lipids did not distinguish fiber types, but clearly separated young, old, and MS subjects. In conclusion, our study suggests that MS in the elderly persons is associated with alterations in skeletal muscle at a fiber-type specific level. Overall, these fiber type-specific modifications may be important both for the age-related loss of muscle mass and strength and for the increased prevalence of MS in elderly subjects.

The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the beta- and gamma-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical betagamma-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the beta-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the betagamma-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINbetagamma clearly interacts with AKINbeta-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINbetagamma gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINbetagamma expression. A two-hybrid screen performed with AKINbetagamma as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINbetagamma and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINbetagamma-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions.