Antibacterial Activity and Mechanism of Action of Bovine Lactoferricin Derivatives with Symmetrical Amino Acid Sequences.

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18⁻28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18⁻28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs.

Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity against Human Cells of a Peptide Derived from Bovine αS1-Casein.

The primary objective of this study was to improve our understanding of the antimicrobial mechanism of protein-derived peptides and to provide evidence for protein-derived peptides as food bio-preservatives by examining the antimicrobial activities, low cytotoxicity, stabilities, and mechanism of Cp1 (LRLKKYKVPQL). In this study, the protein-derived peptide Cp1 was synthesized from bovine αS1-casein, and its potential use as a food biopreservative was indicated by the higher cell selectivity shown by 11-residue peptide towards bacterial cells than human RBCs. It also showed broad-spectrum antimicrobial activity, with minimum inhibitory concentrations (MICs) of 64⁻640 µM against both gram-positive and gram-negative bacteria. The peptide had low hemolytic activity (23.54%, 512 µM) as well as cytotoxicity. The results of fluorescence spectroscopy, flow cytometry, and electron microscopy experiments indicated that Cp1 exerted its activity by permeabilizing the microbial membrane and destroying cell membrane integrity. We found that Cp1 had broad-spectrum antimicrobial activity, low hemolytic activity, and cytotoxicity. The results also revealed that Cp1 could cause cell death by permeabilizing the cell membrane and disrupting membrane integrity. Overall, the findings presented in this study improve our understanding of the antimicrobial potency of Cp1 and provided evidence of the antimicrobial mechanisms of Cp1. The peptide Cp1 could have potential applications as a food biopreservative.

[Study on biological characteristics and stability of the linear derivative Bac2a from bactenecin].

The objective of the study is to analyze the biological characteristics and stability of the linear derivative Bac2a from bactenecin, compared with the control peptide melittin. The secondary structure, antibacterial activity, hemolytic activity, cell toxicity and stability of the Bac2a were determined by circular dichroism spectroscopy, broth micro-dilution method and MTT assay. The results showed that Bac2a was a nonregular curl in aqueous solution, however, it was an α-helix structure in the hydrophobic environment. The minimal inhibitory concentration (MIC) of Bac2a ranged from 2 to 32 µmol/L, so the bacteriostatic activity of Bac2a was strong. The hemolytic rate was only 14.81% when the concentration of Bac2a was 64 µmol/L, which showed that the hemolytic rate of Bac2a was low. The therapy index of Bac2a was 3.26, and the cytotoxicity was relatively low, thus the cell selectivity was relatively high. In addition, with the heating treatment of 100℃ for 1 h, Bac2a still possessed rather a high antibacterial activity and showed a good heating stability. In a word, Bac2a has good application prospects in food, medicine and other fields, and is expected as a substitute for traditional antibiotics.

Chemical enhancement of torsinA function in cell and animal models of torsion dystonia.

Movement disorders represent a significant societal burden for which therapeutic options are limited and focused on treating disease symptomality. Early-onset torsion dystonia (EOTD) is one such disorder characterized by sustained and involuntary muscle contractions that frequently cause repetitive movements or abnormal postures. Transmitted in an autosomal dominant manner with reduced penetrance, EOTD is caused in most cases by the deletion of a glutamic acid (DeltaE) in the DYT1 (also known as TOR1A) gene product, torsinA. Although some patients respond well to anticholingerics, therapy is primarily limited to either neurosurgery or chemodenervation. As mutant torsinA (DeltaE) expression results in decreased torsinA function, therapeutic strategies directed toward enhancement of wild-type (WT) torsinA activity in patients who are heterozygous for mutant DYT1 may restore normal cellular functionality. Here, we report results from the first-ever screen for candidate small molecule therapeutics for EOTD, using multiple activity-based readouts for torsinA function in Caenorhabditis elegans, subsequent validation in human DYT1 patient fibroblasts, and behavioral rescue in a mouse model of DYT1 dystonia. We exploited the nematode to rapidly discern chemical effectors of torsinA and identified two classes of antibiotics, quinolones and aminopenicillins, which enhance WT torsinA activity in two separate in vivo assays. Representative molecules were assayed in EOTD patient fibroblasts for improvements in torsinA-dependent secretory function, which was improved significantly by ampicillin. Furthermore, a behavioral defect associated with an EOTD mouse knock-in model was also rescued following administration of ampicillin. These combined data indicate that specific small molecules that enhance torsinA activity represent a promising new approach toward therapeutic development for EOTD, and potentially for other diseases involving the processing of mutant proteins.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson's disease model.

Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.

Deletion of the ubiquitin ligase CHIP leads to the accumulation, but not the aggregation, of both endogenous phospho- and caspase-3-cleaved tau species.

Accumulation of the microtubule-associated protein tau into neurofibrillary lesions is a pathological consequence of several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Hereditary mutations in the MAPT gene were shown to promote the formation of structurally distinct tau aggregates in patients that had a parkinsonian-like clinical presentation. Whether tau aggregates themselves or the soluble intermediate species that precede their aggregation are neurotoxic entities in these disorders has yet to be resolved; however, recent in vivo evidence supports the latter. We hypothesized that depletion of CHIP, a tau ubiquitin ligase, would lead to an increase in abnormal tau. Here, we show that deletion of CHIP in mice leads to the accumulation of non-aggregated, ubiquitin-negative, hyperphosphorylated tau species. CHIP-/- mice also have increased neuronal caspase-3 levels and activity, as well as caspase-cleaved tau immunoreactivity. Overexpression of mutant (P301L) human tau in CHIP-/- mice is insufficient to promote either argyrophilic or "pre-tangle" structures, despite marked phospho-tau accumulation throughout the brain. These observations are supported in post-developmental studies using RNA interference for CHIP (chn-1) in Caenorhabditis elegans and cell culture systems. Our results demonstrate that CHIP is a primary component in the ubiquitin-dependent degradation of tau. We also show that hyperphosphorylation and caspase-3 cleavage of tau both occur before aggregate formation. Based on these findings, we propose that polyubiquitination of tau by CHIP may facilitate the formation of insoluble filamentous tau lesions.

Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

Torsin-mediated protection from cellular stress in the dopaminergic neurons of Caenorhabditis elegans.

Parkinson's disease (PD) is linked genetically to proteins that function in the management of cellular stress resulting from protein misfolding and oxidative damage. Overexpression or mutation of alpha-synuclein results in the formation of Lewy bodies and neurodegeneration of dopaminergic (DA) neurons. Human torsinA, mutations in which cause another movement disorder termed early-onset torsion dystonia, is highly expressed in DA neurons and is also a component of Lewy bodies. Previous work has established torsins as having molecular chaperone activity. Thus, we examined the ability of torsinA to manage cellular stress within DA neurons of the nematode Caenorhabditis elegans. Worm DA neurons undergo a reproducible pattern of neurodegeneration after treatment with 6-hydroxydopamine (6-OHDA), a neurotoxin commonly used to model PD. Overexpression of torsins in C. elegans DA neurons results in dramatic suppression of neurodegeneration after 6-OHDA treatment. In contrast, expression of either dystonia-associated mutant torsinA or combined overexpression of wild-type and mutant torsinA yielded greatly diminished neuroprotection against 6-OHDA. We further demonstrated that torsins seem to protect DA neurons from 6-OHDA through downregulating protein levels of the dopamine transporter (DAT-1) in vivo. Additionally, we determined that torsins protect robustly against DA neurodegeneration caused by overexpression of alpha-synuclein. Using mutant nematodes lacking DAT-1 function, we also showed that torsin neuroprotection from alpha-synuclein-induced degeneration occurs in a manner independent of this transporter. Together, these data have mechanistic implications for movement disorders, because our results demonstrate that torsin proteins have the capacity to manage sources of cellular stress within DA neurons.

Suppression of polyglutamine-induced protein aggregation in Caenorhabditis elegans by torsin proteins.

Torsion dystonia is an autosomal dominant movement disorder characterized by involuntary, repetitive muscle contractions and twisted postures. The most severe early-onset form of dystonia has been linked to mutations in the human DYT1 (TOR1A) gene encoding a protein termed torsinA. While causative genetic alterations have been identified, the function of torsin proteins and the molecular mechanism underlying dystonia remain unknown. Phylogenetic analysis of the torsin protein family indicates these proteins share distant sequence similarity with the large and diverse family of AAA+ proteins. We have established the nematode, Caenorhabditis elegans, as a model system for examining torsin activity. Using an in vivo assay for polyglutamine repeat-induced protein aggregation in living animals, we have determined that ectopic overexpression of both human and C. elegans torsin proteins results in a dramatic reduction of polyglutamine-dependent protein aggregation in a manner similar to that previously reported for molecular chaperones. The suppressive effects of torsin overexpression persisted as animals aged, whereas a mutant nematode torsin protein was incapable of ameliorating aggregate formation. Antibody staining of transgenic animals indicated that both the C. elegans torsin-related protein TOR-2 and ubiquitin were localized to sites of protein aggregation. These data represent the first functional evidence of a role for torsins in effectively managing protein folding and suggest that possible breakdown in a neuroprotective mechanism that is, in part, mediated by torsins may be responsible for the neuronal dysfunction associated with dystonia.