Engineering of protease variants exhibiting altered substrate specificity.

By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Intracellular cleavage of amyloid ß by a viral protease NIa prevents amyloid ß-mediated cytotoxicity.

Nuclear inclusion a (NIa) of turnip mosaic virus is a cytosolic protease that cleaves amyloid ß (Aß) when heterologously overexpressed. Lentivirus-mediated expression of NIa in the brains of APP(sw)/PS1 mice significantly reduces cerebral Aß levels and plaque depositions, and improves behavioral deficits. Here, the effects of NIa and neprilysin (NEP), a well-known Aß-cleaving protease, on oligomeric Aß-induced cell death were evaluated. NIa cleaved monomeric and oligomeric Aß at a similar rate, whereas NEP only cleaved monomeric Aß. Oligomeric Aß-induced cytotoxicity and mitochondrial dysfunction were significantly ameliorated by NIa, but not by NEP. Endocytosed fluorescently-labeled Aß localized to mitochondria, and this was significantly reduced by NIa, but not by NEP. These data suggest that NIa may exerts its protective roles by degrading Aß and thus preventing mitochondrial deposition of Aß.

Activation of Autophagy Pathway Suppresses the Expression of iNOS, IL6 and Cell Death of LPS-Stimulated Microglia Cells.

Microglia play a role in maintaining and resolving brain tissue homeostasis. In pathological conditions, microglia release pro-inflammatory cytokines and cytotoxic factors, which aggravate the progression of neurodegenerative diseases. Autophagy pathway might be involved in the production of pro-inflammatory cytokines and cytotoxic factors in microglia, though details of the mechanism remain largely unknown. In the present study, we examined the role of the autophagy pathway in activated BV2 microglia cells. In BV2 cells, rapamycin treatment activated the formation of anti-LC3-labeled autophagosomes, whereas the ATG5 depletion using siRNA-ATG5 prevented the formation of LC3-labeled autophagosomes, indicating that BV2 cells exhibit an active classical autophagy system. When treated with LPS, BV2 cells expressed an increase of anti-LC3-labeled dots. The levels of LC3-labeled dots were not suppressed, instead tended to be enhanced, by the inhibition of the autophagy pathway with siRNA-ATG5 or wortmannin, suggesting that LPS-induced LC3-labeled dots in nature were distinct from the typical autophagosomes. The levels of LPS-induced expression of iNOS and IL6 were suppressed by treatment with rapamycin, and conversely, their expressions were enhanced by siRNA-ATG5 treatment. Moreover, the activation of the autophagy pathway using rapamycin inhibited cell death of LPS-stimulated microglia. These results suggest that although microglia possess a typical autophagy pathway, the glial cells express a non-typical autophagy pathway in response to LPS, and the activation of the autophagy pathway suppresses the expression of iNOS and IL6, and the cell death of LPS-stimulated microglia.

Expression of the plant viral protease NIa in the brain of a mouse model of Alzheimer's disease mitigates Aß pathology and improves cognitive function.

The plant viral protease, NIa, has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln, with a scissoring property after Gln. We recently reported that NIa efficiently cleaved the amyloid-ß (Aß) peptide, which contains the sequence Val-His-His-Gln in the vicinity of the cleavage site by α-secretase, and that the expression of NIa using a lentiviral system in the brain of AD mouse model reduced plaque deposition levels. In the present study, we investigated whether exogenous expression of NIa in the brain of AD mouse model is beneficial to the improvement of cognitive deficits. To address this question, Lenti-NIa was intracerebrally injected into the brain of Tg-APPswe/ PS1dE9 (Tg-APP/PS1) mice at 7 months of age and behavioral tests were performed 15-30 days afterwards. The results of the water maze test indicated that Tg-APP/PS1 mice which had been injected with Lenti-GFP showed an increased latency in finding the hidden-platform and markedly enhanced navigation near the maze-wall, and that such behavioral deficits were significantly reversed in Tg-APP/PS1 mice injected with Lenti-NIa. In the passive avoidance test, Tg-APP/PS1 mice exhibited a severe deficit in their contextual memory retention, which was reversed by NIa expression. In the marble burying test, Tg-APP/PS1 mice buried marbles fewer than non-transgenic mice, which was also significantly improved by NIa. After behavioral tests, it was verified that the Tg-APP/PS1 mice with Lenti-NIa injection had reduced Aß levels and plaque deposition when compared to Tg-APP/PS1 mice. These results showed that the plant viral protease, NIa, not only reduces Aß pathology, but also improves behavioral deficits.

An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.

BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.

The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) cleaves amyloid-ß.

BACKGROUND: The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12)-His-His-Gln(15), near the presumptive α-secretase cleavage site of the amyloid-ß (Aß) peptide led us to hypothesize that NIa could possess activity against Aß. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aß can be degraded by NIa in vitro. The specific cleavage of Aß was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aß. Moreover, lentiviral-mediated expression of NIa in APP(sw)/PS1 transgenic mice significantly reduced the levels of Aß and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aß in the cytoplasm could be a novel strategy to control the levels of Aß, plaque formation, and the associated cell death.

Microplate hybridization assay for detection of isoniazid resistance in Mycobacterium tuberculosis.

Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

Engineering of Kex2 variants exhibiting altered substrate specificity.

Engineering of secreted protease variants exhibiting altered substrate specificity is a challenging task because effective screening methods for the desired property are not available yet. In this study, we sought to obtain variants of Kex2, a yeast Golgi protease, which exhibit altered P2 specificity. We first randomly mutated three Asp residues (D176, D210, and D211) that constitute the S2 pocket of Kex2 and then isolated from the resulting library Kex2 variants that preferred substrates with Met (poorly preferred by wild type Kex2) at the P2 position using a yeast-based screening method. The Kex2 variants isolated from this initial screening were further tested against various substrate sequences. Four out of the 16 isolated Kex2 variants showed greater preference for Met than for Lys (preferred by the wild-type Kex2) at the P2 position. We therefore suggest that our method might serve as an efficient tool for engineering and directing the evolution of secreted proteases.