Activity of Chemically Synthesized Peptide Encoded by the miR156A Precursor and Conserved in the Brassicaceae Family Plants.

It was recently found that the primary transcripts of some microRNA genes (pri-miRNAs) are able to express peptides with 12 to 40 residues in length. These peptides, called miPEPs, participate in the transcriptional regulation of their own pri-miRNAs. In our previous studies, we used bioinformatic approach for comparative analysis of pri-miRNA sequences in plant genomes to identify a new group of miPEPs (miPEP-156a peptides) encoded by pri-miR156a in several dozen species of the Brassicaceae family. Exogenous miPEP-156a peptides could efficiently penetrate into the plant seedlings through the root system and spread systemically to the leaves. The peptides produced moderate morphological effect accelerating primary root growth. In parallel, the miPEP-156a peptides upregulated expression of their own pri-miR156a. Importantly, the observed effects at both morphological and molecular levels correlated with the peptide ability to quickly translocate into the cell nucleus and to bind chromatin. In this work, we established secondary structure of the miPEP-156a and demonstrated its changes induced by formation of the peptide complex with DNA.

Glutenase and collagenase activities of wheat cysteine protease Triticain-α: feasibility for enzymatic therapy assays.

Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-α was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-α-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer α-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-α was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-α can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications.

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein.

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the 'S genome' originated from Nicotiana sylvestris and the 'T genome' derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.

The Role of Microtubule Association in Plasmodesmal Targeting of Potato mop-top virus Movement Protein TGBp1.

Cell-to-cell movement of Potato mop-top virus (PMTV) is mediated by three virus-encoded 'triple gene block' (TGB) proteins termed TGBp1, TGBp2 and TGBp3. TGBp1 binds virus RNAs to form viral ribonucleoprotein complexes (vRNPs), the transport form of viral genome. TGBp2 and TGBp3 are necessary for intracellular delivery of TGBp1-containing vRNPs to plasmodesmata. To analyze subcellular localization and transport of TGBp1 we used a single binary vector for agrobacterium-mediated co-expression of PMTV TGBp1 fused to green fluorescent protein and TGBp2/TGBp3. At two days post infiltration (dpi) TGBp1 was found in the nucleus and in association with microtubules (MTs). Similar localization pattern was revealed in cells expressing GFP-TGBp1 alone after particle bombardment. At 3 dpi, in addition to the nucleus and MTs, TGBp1 was detected in numerous granular bodies located both along the MTs and at the cell wall. The latter structures co-localized with plasmodesmata-associated callose depositions. At 4 dpi, GFP-TGBp1 was detected in cell wall-associated bodies and also in residual MTs, the nucleoplasm and large perinuclear inclusions resembling aggresomes. Therefore GFP-TGBp1 association with MTs preceded to its localization to plasmodesmata. Disassembly of cell MTs by colchicine prevented GFP-TGBp1 targeting to plasmodesmata and the MT-dependent aggresome formation. Deletion analysis also revealed a correlation between TGBp1 microtubule association and plasmodesmata targeting. We propose that TGBp1 interaction with MTs may be important for the formation of vRNP bodies destined for the transport to plasmodesmata as well as degradation of the excessive TGBp1.

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking.

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral 'triple gene block' (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.

Varied movement strategies employed by triple gene block-encoding viruses.

Several RNA virus genera belonging to the Virgaviridae and Flexiviridae families encode proteins organized in a triple gene block (TGB) that facilitate cell-to-cell and long-distance movement. The TGB proteins have been traditionally classified as hordei-like or potex-like based on phylogenetic comparisons and differences in movement mechanisms of the Hordeivirus and Potexvirus spp. However, accumulating data from other model viruses suggests that a revised framework is needed to accommodate the profound differences in protein interactions occurring during infection and ancillary capsid protein requirements for movement. The goal of this article is to highlight common features of the TGB proteins and salient differences in movement properties exhibited by individual viruses encoding these proteins. We discuss common and divergent aspects of the TGB transport machinery, describe putative nucleoprotein movement complexes, highlight recent data on TGB protein interactions and topological properties, and review membrane associations occurring during subcellular targeting and cell-to-cell movement. We conclude that the existing models cannot be used to explain all TGB viruses, and we propose provisional Potexvirus, Hordeivirus, and Pomovirus models. We also suggest areas that might profit from future research on viruses harboring this intriguing arrangement of movement proteins.

Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1.

Three 'triple gene block' proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-to-cell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection.

A minimal region in the NTPase/helicase domain of the TGBp1 plant virus movement protein is responsible for ATPase activity and cooperative RNA binding.

The TGBp1 protein, encoded in the genomes of a number of plant virus genera as the first gene of the 'triple gene block', possesses an NTPase/helicase domain characterized by seven conserved sequence motifs. It has been shown that the TGBp1 NTPase/helicase domain exhibits NTPase, RNA helicase and RNA-binding activities. In this paper, we have analysed a series of deletion and point mutants in the TGBp1 proteins encoded by Potato virus X (PVX, genus Potexvirus) and Poa semilatent virus (PSLV, genus Hordeivirus) to map functional regions responsible for their biochemical activities in vitro. It was found that, in both PVX and PSLV, the N-terminal part of the TGBp1 NTPase/helicase domain comprising conserved motifs I, Ia and II was sufficient for ATP hydrolysis, RNA binding and homologous protein-protein interactions. Point mutations in a single conserved basic amino acid residue upstream of motif I had little effect on the activities of C-terminally truncated mutants of both TGBp1 proteins. However, when introduced into the full-length NTPase/helicase domains, these mutations caused a substantial decrease in the ATPase activity of the protein, suggesting that the conserved basic amino acid residue upstream of motif I was required to maintain a reaction-competent conformation of the TGBp1 ATPase active site.

At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 colocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

Assessment of the integral membrane protein topology in living cells.

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.

Coat proteins of two filamentous plant viruses display NTPase activity in vitro.

Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity.

Triple gene block: modular design of a multifunctional machine for plant virus movement.

Many plant virus genera encode a 'triple gene block' (TGB), a specialized evolutionarily conserved gene module involved in the cell-to-cell and long-distance movement of viruses. The TGB-based transport system exploits the co-ordinated action of three polypeptides to deliver viral genomes to plasmodesmata and to accomplish virus entry into neighbouring cells. Although data obtained on both the TGB and well-studied single protein transport systems clearly demonstrate that plant viruses employ host cell pathways for intra- and intercellular trafficking of genomic nucleic acids and proteins, there is no integral picture of the details of molecular events during TGB-mediated virus movement. Undoubtedly, understanding the molecular basis of the concerted action of TGB-encoded proteins in transporting viral genomes from cell to cell should provide new insights into the general principles of movement protein function. This review describes the structure, phylogeny and expression of TGB proteins, their roles in virus cell-to-cell movement and potential influence on host antiviral defences.

Activities associated with the putative replication initiation protein of coconut foliar decay virus, a tentative member of the genus Nanovirus.

The putative replication initiation protein (Rep) of Coconut foliar decay virus (CFDV) was expressed as a 6x His recombinant protein in E. coli and in recombinant baculovirus. Purified 6x His-Rep protein was demonstrated to possess sequence non-specific RNA- and ssDNA-binding activities as well as magnesium-dependent ATPase/GTPase activity. The yeast two-hybrid system revealed that CFDV Rep could interact with itself. Subcellular distribution of the CFDV Rep was studied by fractionation of insect cells infected with recombinant baculovirus expressing the 6x His-Rep protein and by laser scanning confocal microscopy of Nicotiana benthamiana epidermal cells bombarded with a construct encoding CFDV Rep fused to GFP. It was shown that CFDV Rep associated predominantly with nuclei and membranes of infected/transfected cells. These activities of CFDV-encoded Rep are very similar to those reported for Reps of geminiviruses.