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.

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date,the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase,adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays,colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing N-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 co-target an internal degron of the Mgt1 DNA repair protein. Ufd4 is an E3 of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we report that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately over two decades.

Objective To evaluate changes in peripapillary and macular choroidal thickness and volume after the water-drinking test (WDT) using swept-source optical coherence tomography (SS-OCT). Design Prospective, cross-sectional observational study. Participants Fifty-six eyes of 28 healthy volunteers. Methods Participants underwent a 3-dimensional optic disc and macula scanning protocol with a prototype SS-OCT (Topcon Inc., Tokyo, Japan) at baseline and 15, 30, 45, and 120 minutes after the start of the WDT. The WDT consisted of drinking 1000mL of water within five minutes. Objective measurements of the choroid were obtained with automated segmentation of the choroidal boundaries. Main Outcome Measures Choroidal thickness and volume. Results Mean (standard deviation) age of participants was 35.6 ± 9.1 years. Intraocular pressure (IOP) increased from 14.9 ± 2.7 mmHg at baseline to a peak of 16.8 ± 3.0 mmHg at 15 minutes after the WDT (p<0.001). Mean baseline choroidal thickness and volume were 181.3 ± 50.8 μm and 6.19 ± 1.80 mm3 at the optic disc and 217.4 ± 43.6 μm and 7.83 ± 1.55 mm3 at the macula. Following the WDT, peripapillary and macular choroidal thickness increased by a maximum of 5.7% (P < 0.001) and 4.3% (P < 0.001) respectively. Choroidal volumes increased by 6.4% (P < 0.001) and 3.9% (P < 0.001), respectively. There was no association between change in IOP and peripapillary (P = 0.27) or macular (P = 0.09) choroidal thickness. Conclusions Using automated segmentation of SS-OCT measurements, significant increases in choroidal thickness and volume are observed after the WDT in healthy subjects.

Myomesin is a structural component of the M-band that is expressed in all types of striated muscle. Its primary function may be the maintenance of the thick filament lattice and its anchoring to the elastic filament system composed of titin. Different myomesin isoforms have been described in chicken and mice, but no particular function has been assigned to them. Here we investigate the spatio-temporal expression pattern of myomesin isoforms by means of reverse transcriptasepolymerase chain reaction and isoform-specific antibodies. We find that two alternative splicing events give rise to four myomesin isoforms in chicken contrary to only one splicing event with two possible isoforms in mice. A splicing event at the C terminus results in two splice variants termed H-myomesin and S-myomesin, which represent the major myomesin species in heart and skeletal muscle of avian species, respectively. In contrast, in mammalian heart and skeletal muscle only S-myomesin is expressed. In embryonic heart of birds and mammals, alternative splicing in the central part of the molecule gives rise to the isoform that we termed EH-myomesin. It represents the major myomesin isoform at early embryonic stages of heart but is rapidly down-regulated around birth. Thus, the strict developmental regulation of the EH-myomesin makes it an ideally suited marker for embryonic heart.