A new protein curbs the hypertrophic effect of myostatin inhibition, adding remarkable endurance to motor performance in mice

Current efforts to improve muscle performance are focused on muscle trophism via inhibition of the myostatin pathway: however they have been unsuccessful in the clinic to date. In this study, a novel protein has been created by combining the soluble activin receptor, a strong myostatin inhibitor, to the C-terminal agrin nLG3 domain (ActR-Fc-nLG3) involved in the development and maintenance of neuromuscular junctions. Both domains are connected via the constant region of an Igg1 monoclonal antibody. Surprisingly, young male mice treated with ActR-Fc-nLG3 showed a remarkably increased endurance in the rotarod test, significantly longer than the single domain compounds ActR-Fc and Fc-nLG3 treated animals. This increase in endurance was accompanied by only a moderate increase in body weights and wet muscle weights of ActR-Fc-nLG3 treated animals and were lower than expected. The myostatin inhibitor ActR-Fc induced, as expected, a highly significant increase in body and muscle weights compared to control animals and ActR-Fc-nLG3 treated animals. Moreover, the prolonged endurance effect was not observed when ActR-Fc and Fc-nLG3 were dosed simultaneously as a mixture and the body and muscle weights of these animals were very similar to ActR-Fc treated animals, indicating that both domains need to be on one molecule. Muscle morphology induced by ActR-Fc-nLG3 did not appear to be changed however, close examination of the neuromuscular junction showed significantly increased acetylcholine receptor surface area for ActR-Fc-nLG3 treated animals compared to controls. This result is consistent with published observations that endurance training in rats increased acetylcholine receptor quantity at neuromuscular junctions and provide evidence that improving nerve-muscle interaction could be an important factor for sustaining long term muscle activity

Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase

The cytoplasmic coenzyme NAD+-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C1-C4 primary alcohols is a decameric protein with 1 Zn2+-ion and 1–2 Mg2+-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg2+-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD+-dependent activity of MDH with C1–C4 primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg2+-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD+ ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

Towards Random Polypeptide Synthesis

Modern naturally occurring proteins have been produced by a lengthy selective evolutionary process. While, in general, they are all composed of the same 20 amino acids there is a distinct bias in their average amino acid composition. This bias may have arisen due to evolutionary mechanisms, the degeneracy of the genetic code, the primordial availability of suitable monomers, their relative reactivity or a number of other, equally speculative, causes. Mathematics appears to dictate that Nature could not have sampled all possible amino acid sequences and selected the most suitable for a particular function, suggesting that the proteins observed today may have evolved from a relatively small number of precursors. If this is true it would imply that there is a vast set of possible proteins that have simply never existed and that may possess interesting or useful properties. This article investigates whether the structural space occupied by proteins that do not currently exist can be sampled. One approach suggests itself – random polypeptide synthesis in which all possible residue types are inserted at all possible positions of an amino acid sequence of a given length. It is abundantly clear that the truly random synthesis of even a small set of such protein sequences is precluded by simple mathematics. The issues that this raises are discussed and different practical approaches to the problem described

Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders

NMJ morphology of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Confocal images of the NMJs in <i>soleus</i> muscle of P30 Control or SARCO mice, treated or not treated with NT-1654 as indicated. The postsynaptic AChRs were stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was stained with anti-neurofilament and synaptophysin antibodies (NF-Syn; green). The postsynapses were highly fragmented, partially or completely lost in <i>soleus</i> muscle of SARCO mice. The postsynapses of NT-1654 treated SARCO mice resembled those in Control mice, showing pretzel like structures and much less fragmentation. Scale bar: 50 µm. <b>B:</b> The postsynapses in Control mice showed fragmentation class 0 (no fragmentation, for illustration see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088739#pone.0088739.s003" target="_blank">figure S3</a>). The postsynapses of NT-1654 treated SARCO mice were mostly classified into class 1, whereas the postsynapse of SARCO mice showed class 1-4 with a peak at class 3. <b>C:</b> Many NMJs of SARCO mice have terminal nerve sprouting, which was dramatically reduced in the NT1654 treated SARCO mice. Data present mean ± standard deviation, n  =  2 mice, 100 NMJs were counted in each mouse.</p

Fiber type distribution of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Consecutive muscle cross sections of <i>soleus</i> stained with myosine heavy chain (MHC) specific antibodies or cytochrome C oxidase (COX) staining as indicated. Control and treated SARCO mice show clearly separated type I and II fibers. SARCO mice have a significantly increased amount of hybrid fibers (indicated with asterisks). Cytochrome C staining of <i>soleus</i> sections shows a massive reduction of reactivity in SARCO mice which is reverted back to WT levels in treated SARCO mice. <b>B:</b> Quantitative analysis of muscle fibers of <i>soleus.</i> Bars represent mean values ± standard deviation. Control and treated SARCO mice have almost no hybrid fibers and are indistinguishable from each other. Compared to treated ones, SARCO mice have significantly increased hybrid fibers. Type I fibers are significantly decreased. The total fiber number in SARCO mice is also significantly decreased compared to treated animals. Number of animals: wt  =  3; SARCO treated  =  5, SARCO  =  3. * p<0.05; ** p<0.01. Scale bar: 100 µm.</p