Adeno-Associated Virus VP1u Exhibits Protease Activity.

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.

Short Aß peptides attenuate Aß42 toxicity in vivo.

Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.

Characteristics of candidate genes associated with embryonic development in the cow: Evidence for a role for WBP1 in development to the blastocyst stage.

The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (BRINP3, C1QB, HSPA1L, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3 and WBP1) participate in embryonic development. Gene expression was evaluated in matured oocytes and embryos. BRINP3 and C1QB were not detected at any stage. For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage. Exceptions were for PARM1 and WBP1, where steady-state mRNA increased at the 9-16 cell stage. The SNP in WBP1 caused large differences in the predicted three-dimensional structure of the protein while the SNP in PARM1 caused smaller changes. The mutation in WBP1 causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions. Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution. Knockdown of mRNA for WBP1 decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts. WBP1 is an important gene for embryonic development in the cow. Further research to identify how the SNP in WBP1 affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted.

The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis.

Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

Cryoannealing-induced space-group transition of crystals of the carbonic anhydrase psCA3.

Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the ß-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P21212 and was scaled to 2.6 Šresolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Šin resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor.

Microgels with tunable affinity-controlled protein release via desolvation of self-assembled peptide nanofibers.

With a growing number of bioactive protein drugs approved for clinical use each year, there is increasing need for vehicles for localized protein delivery to reduce administered doses, prevent off-target activity, and maintain protein bioactivity. Ideal protein delivery vehicles provide high encapsulation efficiency of bioactive drug, enable fine-tuning of protein release profiles, are biocompatible, and can be administered via minimally-invasive routes. Here we developed an approach to create micron-sized hydrated gels (i.e."microgels") for protein delivery that fulfill these requirements via desolvation of self-assembled ß-sheet peptide nanofibers. Specifically, aqueous solutions of peptide nanofibers were diluted under stirring conditions in a "desolvating agent", such as ethanol, which is miscible with water but poorly solvates peptides. The desolvating agent induced nanofiber physical crosslinking into microgels that retained ß-sheet secondary structure and were stable in aqueous solutions. Microgels did not activate dendritic cells in vitro, suggesting they are biocompatible. Peptide nanofibers and proteins having similar non-solvent immiscibility properties were co-desolvated to produce protein-loaded microgels with loading efficiencies of ∼85%. Encapsulated bioactive proteins rapidly diffused into bulk aqueous media, as expected for hydrated gels. Modifying peptide nanofibers with a protein-binding ligand provided tunable affinity-controlled protein release. Biocompatible microgels formed via desolvation of self-assembled peptide nanofibers are therefore likely to be broadly useful as vehicles for localized delivery of bioactive proteins, as well as other therapeutic molecules.