Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

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.

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.

Anti-HIV activity and resistance profile of the CXC chemokine receptor 4 antagonist POL3026.

We have studied the mechanism of action of Arg(*)-Arg-Nal(2)-Cys(1x)-Tyr-Gln-Lys-(d-Pro)-Pro-Tyr-Arg-Cit-Cys(1x)-Arg-Gly-(d-Pro)(*) (POL3026), a novel specific beta-hairpin mimetic CXC chemokine receptor (CXCR)4 antagonist. POL3026 specifically blocked the binding of anti-CXCR4 monoclonal antibody 12G5 and the intracellular Ca(2+) signal induced by CXC chemokine ligand 12. POL3026 consistently blocked the replication of human immunodeficiency virus (HIV), including a wide panel of X4 and dualtropic strains and subtypes in several culture models, with 50% effective concentrations (EC(50)) at the subnanomolar range, making POL3026 the most potent CXCR4 antagonist described to date. However, 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)-resistant and stromal cell-derived factor-1alpha-resistant HIV-1 strains were cross-resistant to POL3026. Time of addition experiments and a multiparametric evaluation of HIV envelope function in the presence of test compounds confirmed the activity of POL3026 at an early step of virus replication: interaction with the coreceptor. Generation of HIV-1 resistance to POL3026 led to the selection of viruses 12- and 25-fold less sensitive and with mutations in gp120, including the V3 loop region. However, POL3026 prevented the emergence of CXCR4-using variants from an R5 HIV-1 strain that may occur in the presence of anti-HIV agents targeting CC chemokine receptor 5.

Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage.

This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display.

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.

Properties and structure-activity studies of cyclic beta-hairpin peptidomimetics based on the cationic antimicrobial peptide protegrin I.

The properties and structure-activity relationships (SAR) of a macrocyclic analogue of porcine protegrin I (PG-I) have been investigated. The lead compound, having the sequence cyclo-(-Leu-Arg-Leu-Lys-Lys-Arg-Arg-Trp-Lys-Tyr-Arg-Val-d-Pro-Pro-), shows antimicrobial activity against Gram-positive and -negative bacteria, but a much lower haemolytic activity and a much reduced ability to induce dye release from phosphatidylcholine/phosphatidylglycerol liposomes, when compared to PG-I. The enantiomeric form of the lead peptide shows comparable antimicrobial activity, a property shared with other cationic antimicrobial peptides acting on cell membranes. SAR studies involving the synthesis and biological profiling of over 100 single site substituted analogues, showed that the antimicrobial activity was tolerant to a large number of the substitutions tested. Some analogues showed slightly improved antimicrobial activities (2-4-fold lowering of MICs), whereas other substitutions caused large increases in haemolytic activity on human red blood cells.

A family of macrocyclic antibiotics with a mixed peptide-peptoid beta-hairpin backbone conformation.

Macrocyclic peptidomimetics having a mixed peptide-peptoid backbone have been synthesized and shown to possess antibiotic activity against gram-positive and -negative bacteria with a low hemolytic activity against human erythrocytes; one is shown to adopt a regular beta-hairpin conformation by NMR in aqueous solution.

Macrocyclic hairpin mimetics of the cationic antimicrobial peptide protegrin I: a new family of broad-spectrum antibiotics.

The problems associated with increasing antibiotic resistance have stimulated great interest in newly discovered families of naturally occurring cationic antimicrobial peptides. These include protegrin, tachyplesin, and RTD-1, which adopt beta-hairpin-like structures. We report here an approach to novel peptidomimetics based on these natural products. The mimetics were designed by transplanting the cationic and hydrophobic residues onto a beta-hairpin-inducing template, either a D-Pro-L-Pro dipeptide or a xanthene derivative. The mimetics have good antimicrobial activity against Gram-positive and Gram-negative bacteria (minimal inhibitory concentration approximately 6-25 microgram mL(-1)). Analogues with improved selectivity for microbial rather than red blood cells (1 % hemolysis at 100 microgram mL(-1)) were identified from a small library prepared by parallel synthesis. Thus, it is possible to separate the antimicrobial and hemolytic activities in this class of mimetics. NMR studies on one mimetic revealed a largely unordered structure in water, but a transition to a regular beta-hairpin backbone conformation in the presence of dodecylphosphocholine micelles. This family of mimetics may provide a starting point for the optimization of antimicrobial agents of potential clinical value in the fight against multiple-drug-resistant microorganisms.

Crystal structure of OxyB, a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis.

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-A resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys(347) being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.

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 C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-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 C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-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.