PPIases Par14/Par17 Affect HBV Replication in Multiple Ways.

Human parvulin 14 (Par14) and parvulin 17 (Par17) are peptidyl-prolyl cis/trans isomerases that upregulate hepatitis B virus (HBV) replication by binding to the conserved 133Arg-Pro134 (RP) motif of HBc and core particles, and 19RP20-28RP29 motifs of HBx. In the absence of HBx, Par14/Par17 have no effect on HBV replication. Interaction with Par14/Par17 enhances the stability of HBx, core particles, and HBc. Par14/Par17 binds outside and inside core particles and is involved in HBc dimer-dimer interaction to facilitate core particle assembly. Although HBc RP motif is important for HBV replication, R133 residue is solely important for its interaction with Par14/Par17. Interaction of Par14 and Par17 with HBx involves two substrate-binding residues, Glu46/Asp74 (E46/D74) and E71/D99, respectively, and promotes HBx translocation to the nucleus and mitochondria. In the presence of HBx, Par14/Par17 are efficiently recruited to cccDNA and promote transcriptional activation via specific DNA-binding residues Ser19/44 (S19/44). S19 and E46/D74 of Par14, and S44 and E71/D99 of Par17, are also involved in the recruitment of HBc onto cccDNA. Par14/Par17 upregulate HBV replication via various effects that are mediated in part through the HBx-Par14/Par17-cccDNA complex and triple HBc, Par14/Par17, and cccDNA interactions in the nucleus, as well as via core particle-Par14/Par17 interactions in the cytoplasm.

Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17.

Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.

The Multiple Roles of Peptidyl Prolyl Isomerases in Brain Cancer.

Peptidyl prolyl isomerases (PPIases) are broadly expressed enzymes that accelerate the cis-trans isomerization of proline peptide bonds. The most extensively studied PPIase family member is protein interacting with never in mitosis A1 (PIN1), which isomerizes phosphorylated serine/threonine⁻proline bonds. By catalyzing this specific cis-trans isomerization, PIN1 can alter the structure of its target proteins and modulate their activities in a number of different ways. Many proteins are targets of proline-directed phosphorylation and thus PIN1-mediated isomerization of proline bonds represents an important step in the regulation of a variety of cellular mechanisms. Numerous other proteins in addition to PIN1 are endowed with PPIase activity. These include other members of the parvulin family to which PIN1 belongs, such as PIN4, as well as several cyclophilins and FK506-binding proteins. Unlike PIN1, however, these other PPIases do not isomerize phosphorylated serine/threonine⁻proline bonds and have different substrate specificities. PIN1 and other PPIases are overexpressed in many types of cancer and have been implicated in various oncogenic processes. This review will discuss studies providing evidence for multiple roles of PIN1 and other PPIases in glioblastoma and medulloblastoma, the most frequent adult and pediatric primary brain tumors.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.