The TPR- and J-domain-containing proteins DJC31 and DJC62 are involved in abiotic stress responses in Arabidopsis thaliana

ABSTRACT Molecular chaperones play an important role during the response to different stresses. Since plants are sessile organisms, they need to be able to adapt quickly to different conditions. To do so, plants possess a complex chaperone machinery, composed of HSP70, HSP90, J proteins and other factors. In this study we characterized DJC31 (also known as TPR16) and DJC62 (also known as TPR15) of Arabidopsis thaliana, two J proteins that additionally carry clamp-type tetratricopeptide repeat domains. Using cell fractionation and split GFP, we could show that both proteins are attached to the cytosolic side of the endoplasmic reticulum membrane. Moreover, an interaction with cytosolic HSP70.1 and HSP90.2 could be shown using bimolecular fluorescence complementation. Knockout of both DJC31 and DJC62 caused severe defects in growth and development, which affected almost all organs. Furthermore, it could be shown that the double mutant is more sensitive to osmotic stress and treatment with abscisic acid, but surprisingly exhibited enhanced tolerance to drought. Taken together, these findings indicate that DJC31 and DJC62 might act as important regulators of chaperone-dependent signaling pathways involved in plant development and stress responses.

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and independent pathways

In angiosperms, the α/β hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely non-canonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (−)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity towards (−)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. By contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the øX174 Capsid

Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging is specific to single-stranded (ss) DNA viruses. ssDNA genome packaging combines elements found in both double-stranded (ds) DNA and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. øX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a øXG4J chimera, in which the øX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, øXα3J. Unlike øXG4J, øXα3J efficiently packaged DNA but produced non-infectious particles. These particles displayed a reduced ability to attach to host cells, suggesting internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and post-packaging phenomena. Importance ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit øX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface.

Deciphering Network Crosstalk: The Current Status and Potential of miRNA Regulatory Networks on the HSP40 Molecular Chaperone Network

Molecular chaperone networks fulfill complex roles in protein homeostasis and are essential for maintaining cell health. Hsp40s (commonly referred to as J-proteins) have critical roles in development and are associated with a variety of human diseases, yet little is known regarding the J-proteins with respect to the post-transcriptional mechanisms that regulate their expression. With relatively small alterations in their abundance and stoichiometry altering their activity, post-transcriptional regulation potentially has significant impact on the functions of J-proteins. MicroRNAs (miRNAs) are a large group of non-coding RNAs that form a complex regulatory network impacting gene expression. Here we review and investigate the current knowledge and potential intersection of miRNA regulatory networks with the J-Protein chaperone network. Analysis of datasets from the current version of TargetScan revealed a great number of predicted microRNAs targeting J-proteins compared to the limited reports of interactions to date. There are likely unstudied regulatory interactions that influence chaperone biology contained within our analysis. We go on to present some criteria for prioritizing candidate interactions including potential cooperative targeting of J-Proteins by multiple miRNAs. In summary, we offer a view on the scope of regulation of J-Proteins through miRNAs with the aim of guiding future investigations by identifying key regulatory nodes within these two complex cellular networks.

Genome-wide identification and expression analysis of dirigent-jacalin genes from plant chimeric lectins in Moso bamboo (Phyllostachys edulis)

Dirigent-jacalin (D-J) genes belong to the plant chimeric lectin family, and play vital roles in plant growth and resistance to abiotic and biotic stresses. To explore the functions of the D-J family in the growth and development of Moso bamboo (Phyllostachys edulis), their physicochemical properties, phylogenetic relationships, gene and protein structures, and expression patterns were analyzed in detail. Four putative PeD-J genes were identified in the Moso bamboo genome, and microsynteny and phylogenetic analyses indicated that they represent a new branch in the evolution of plant lectins. PeD-J proteins were found to be composed of a dirigent domain and a jacalin-related lectin domain, each of which contained two different motifs. Multiple sequence alignment and homologous modeling analysis indicated that the three-dimensional structure of the PeD-J proteins was significantly different compared to other plant lectins, primarily due to the tandem dirigent and jacalin domains. We surveyed the upstream putative promoter regions of the PeD-Js and found that they mainly contained cis-acting elements related to hormone and abiotic stress response. An analysis of the expression patterns of root, leaf, rhizome and panicle revealed that four PeD-J genes were highly expressed in the panicle, indicating that they may be required during the formation and development of several different tissue types in Moso bamboo. Moreover, PeD-J genes were shown to be involved in the rapid growth and development of bamboo shoots. Quantitative Real-time PCR (qRT PCR) assays further verified that D-J family genes were responsive to hormones and stresses. The results of this study will help to elucidate the biological functions of PeD-Js during bamboo growth, development and stress response.

Ubqln4 Facilitates Endoplasmic Reticulum-to-Cytosol Escape of a Nonenveloped Virus during Infection

ABSTRACT The nonenveloped polyomavirus simian virus 40 (SV40) must penetrate the host endoplasmic reticulum (ER) membrane to enter the cytosol in order to promote infection. How this is accomplished is not entirely clear. Here, we demonstrate that the cytosolic chaperone Ubiquilin4 (Ubqln4) binds directly to the ER membrane J proteins B12 and B14. Strategically localized at the ER-cytosol interface, Ubqln4 captures SV40 emerging from the ER, thereby facilitating escape of the virus from the ER into the cytosol, which leads to infection. Strikingly, Ubqln4 engages the J proteins in a J-domain-independent manner, in contrast to the previously reported Hsc70-Hsp105-SGTA-Bag2 cytosolic complex that also mediates SV40 ER-to-cytosol transport. Our results also reveal that the H domain and STI1 motif (1-2) of Ubqln4 support J protein binding, essential for SV40 infection. Together, these data further clarify the molecular basis by which a nonenveloped virus escapes a host membrane during infectious entry. IMPORTANCE How a nonenveloped virus escapes from a host membrane to promote infection remains enigmatic. In the case of the nonenveloped polyomavirus SV40, penetration of the ER membrane to reach the cytosol is a decisive virus infection step. In this study, we found a new host factor called Ubqln4 that facilitates escape of SV40 from the ER into the cytosol, thereby providing a path for the virus to enter the nucleus to cause infection.

The Herpes Simplex Virus 1 Immediate Early Protein ICP22 Is a Functional Mimic of a Cellular J Protein

ABSTRACT Molecular chaperones and cochaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that herpes simplex virus 1 (HSV-1) infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (Virus Induced Chaperone Enriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. Here, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded cochaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of nonnative proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins, and (iii) thermoprotection against heat inactivation of firefly luciferase, and (iv) sequence homology analysis indicated that ICP22 contains an N-terminal J domain and a C-terminal substrate binding domain, similar to type II cellular J proteins. ICP22 may thus be functionally similar to J-protein/Hsp40 cochaperones that function together with their HSP70 partners to prevent aggregation of nonnative proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, since simian immunodeficiency virus T antigen was previously shown to contain a J domain; however, this the first known example of the acquisition of a functional J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J proteins to evolve a multifunctional cochaperone that functions with Hsc70 to promote lytic infection. IMPORTANCE Viruses have evolved a variety of strategies to succeed in a hostile environment. The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 plays several roles in the virus life cycle, including downregulation of cellular gene expression, upregulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of nonnative proteins, and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 functionally resembles a cellular J-protein/HSP40 family cochaperone, interacting specifically with Hsc70. We suggest that HSV has taken advantage of the adaptable nature of J proteins to evolve a multifunctional cochaperone that functions with Hsc70 to promote lytic infection.

The HSV-1 immediate early protein ICP22 is a J-like protein required for Hsc70 reorganization during lytic infection

ABSTRACTMolecular chaperones and co-chaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that HSV-1 infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (VirusInducedChaperoneEnriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. In this paper, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded co-chaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of non-native proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins and (iii) thermoprotection against heat-inactivation of firefly luciferase. (iv) Sequence homology analysis indicated that ICP22 contains an N-terminal J-domain and a C-terminal substrate binding domain, similar to type II cellular J-proteins. ICP22 may, thus, be functionally similar to J-protein/Hsp40 co-chaperones that function together with their HSP70 partners to prevent aggregation of non-native proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, as SV40 T Antigen was previously shown to contain a J-domain; however, this the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J-proteins to evolve a multi-functional co-chaperone that functions with Hsc70 to promote lytic infection.IMPORTANCEViruses have evolved a variety of strategies to succeed in a hostile environment. The HSV immediate early protein ICP22 plays several roles in the virus life cycle including down-regulation of cellular gene expression, up-regulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of non-native proteins and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 resembles a cellular J-protein/HSP40 family co-chaperone, interacting specifically with Hsc70. This is the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has evolved to manipulate the host proteostatic machinery during the establishment of lytic infection.