Structural transitions modulate the chaperone activities of Grp94.

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress.

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.

Lymphocyte soluble factors from pregnant cows modulate mRNA transcript abundances encoding for proteins associated with trophoblast growth and development.

This study was conducted to determine whether T cell populations are responsible for modulating placental development during gestation in cattle. It was hypothesized that CD4+CD25+ and γ/δ+ T cells modulate gene expression, based on mRNA transcript abundances, and promote proliferation and survival of trophoblast cells. Peripheral blood was collected from cows at 160 to 180 days of gestation and non-pregnant cows, T cell populations CD8+, CD4+, CD4+CD25+, CD24+CD25-, and γ/δ+ T cells were isolated, cultured for 48 h, and supernatant was collected. Placental samples were digested, and trophoblast cells were cultured for 24 h. Trophoblast cells were cultured with 50 µL of T cell-conditioned media and 50 µL of fresh culture media for an additional 48 h. Samples in control wells were treated with unconditioned media. Trophoblast cell proliferation, apoptosis, and mRNA transcript assays were conducted. There was no effect of T cell population on trophoblast apoptosis rate, proliferation, and relative mRNA transcript abundances. The T cell supernatant from pregnant and non-pregnant cows induced greater apoptosis rates in trophoblast cells than unconditioned media. Trophoblast cells proliferated less when treated with T cell supernatant from pregnant compared to unconditioned medium and non-pregnant cows. Treatment with the T cell supernatant from pregnant cows resulted in larger abundances of BMP5, IGF1R, PAG10, FGF2, RSPO3 and TMED2 and also a lesser abundance of FGF2 mRNA transcript than non-pregnant group and unconditioned media treatments. Supernatant from T cell derived from pregnant cows modulates trophoblast mRNA transcript abundances differently from T cell supernatant of non-pregnant cows.

Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model.

Zika virus (ZIKV) is currently undergoing pandemic emergence. While disease is typically subclinical, severe neurologic manifestations in fetuses and newborns after congenital infection underscore an urgent need for antiviral interventions. The adenosine analog BCX4430 has broad-spectrum activity against a wide range of RNA viruses, including potent in vivo activity against yellow fever, Marburg and Ebola viruses. We tested this compound against African and Asian lineage ZIKV in cytopathic effect inhibition and virus yield reduction assays in various cell lines. To further evaluate the efficacy in a relevant animal model, we developed a mouse model of severe ZIKV infection, which recapitulates various human disease manifestations including peripheral virus replication, conjunctivitis, encephalitis and myelitis. Time-course quantification of viral RNA accumulation demonstrated robust viral replication in several relevant tissues, including high and persistent viral loads observed in the brain and testis. The presence of viral RNA in various tissues was confirmed by an infectious culture assay as well as immunohistochemical staining of tissue sections. Treatment of ZIKV-infected mice with BCX4430 significantly improved outcome even when treatment was initiated during the peak of viremia. The demonstration of potent activity of BCX4430 against ZIKV in a lethal mouse model warrant its continued clinical development.