FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation

Various mutant lysozymes were constructed by genetic modification and secreted in yeast expression system to evaluate the changes in the antigenicity of hen egg lysozyme (HEL). Although Arg68, the most critical residue to antigenicity of HEL, was substituted with Gln, the binding of monoclonal antibodies (mAbs) with the mutant lysozyme did not critically reduce, remaining 60% of the binding with mAb. In contrast, glycosylated mutant lysozyme G49N whose glycine was substituted with asparagine dramatically reduced the binding with mAb. The oligomannosyl type of G49N lysozyme reduced binding with mAb to one-fifth, while the polymannosyl type of G49N lysozyme completely diminished the binding with mAb. This suggests that the site-specific glycosylation of lysozyme in the interfacial region of lysozyme-antibody complex is more effective to reduce the antigenicity than the mutation of single amino acid substitution in the interfacial region.     

Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.     

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution

Background

The accessory gene regulator (agr) is a quorum sensing cluster of genes which control colonization and virulence in Staphylococcus aureus. We evaluated agr function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against agr functional and dysfunctional HA-MRSA and CA-MRSA.

Methods

40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of agr function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10⁶ and 10⁸ cfu/ml of agr functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model.

Results

15% isolates were agr dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10⁶ cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among agr functional and dysfunctional strains. However, against a high initial inoculum of 10⁸ cfu/ml, killing activity was notably attenuated against agr dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log₁₀ CFU/ml for agr functional vs. 2.41 log₁₀ CFU/ml for agr dysfunctional MRSA (p = 0.0007).

Conclusions

Dysfunction in agr was less common among CA-MRSA vs. HA-MRSA. agr dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.     

Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.     

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Improvement in spermatogenic function after subcutaneous implantation of a capsule containing an aromatase inhibitor in four oligozoospermic dogs and one azoospermic dog with high plasma estradiol-17beta concentrations

A capsule containing an aromatase inhibitor (4-androsten-4-ol-3,17-dione) was subcutaneously implanted in four oligozoospermic beagle dogs and one azoospermic beagle dog with high plasma estradiol-17beta (E2) concentrations (15-19 pg/ml) and low plasma testosterone (T) concentrations (0.6-0.8 ng/ml) for 8 weeks and the effect of the aromatase inhibitor on spermatogenic dysfunction was assessed. Plasma E2 and T concentrations and semen quality were examined at 1 week intervals from 3 weeks before to 12 weeks after the start of treatment. Testicular biopsies were done twice (capsule implantation and removal). Plasma E2 concentrations of all dogs decreased (9-14 pg/ml) and plasma T concentrations increased (2.0-2.6 ng/ml) from 3 weeks after capsule implantation to capsule removal. The mean number of spermatozoa ejaculated by all four oligozoospermic dogs between 4 and 9 weeks after implantation was higher (127 x 10(6) to 205 x 10(6)) than before implantation (20 x 10(6) to 38 x 10(6)) (P < 0.05 and 0.01). Very low numbers (2 x 10(4) to 4 x 10(4)) of immotile spermatozoa were observed between 7 and 8 weeks after implantation in the semen collected from the dog with azoospermia. Before implantation, a few spermatozoa were seen in only one-fifth of the seminiferous tubules in this dog; 8 weeks after implantation, the mean diameter and mean number of round spermatids in the seminiferous tubules in all five dogs were higher than before implantation (P < 0.05). Implantation of the capsule containing the aromatase inhibitor in infertile dogs with abnormally high plasma E2 concentrations improved their spermatogenic function, concurrent with decreased plasma E2 and increased plasma T.     

Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.