Comparison of the folding processes of T. thermophilus and E. coli ribonucleases H

In order to examine how the stabilization of thermophilic proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange. Like its homolog from Escherichia coli, this thermophilic protein populates a partially folded kinetic intermediate within the first few milliseconds of folding. The structure of this intermediate is similar to that of E.coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the protein. Proline isomerization appears to partly limit the folding of the thermophilic but not the mesophilic protein. Lastly, unlike other thermophilic proteins, which unfold much more slowly than their mesophilic counterparts, T.thermophilus RNase H folds and unfolds with overall rates similar to those of E.coli RNase H.     

Cytoplasmic organelles on the road to mRNA decay

Localization of both mRNAs and mRNA decay factors to internal membranes of eukaryotic cells provides a means of coordinately regulating mRNAs with common functions as well as coupling organelle function to mRNA turnover. The classic mechanism of mRNA localization to membranes is the signal sequence-dependent targeting of mRNAs encoding membrane and secreted proteins to the cytoplasmic surface of the endoplasmic reticulum. More recently, however, mRNAs encoding proteins with cytosolic or nuclear functions have been found associated with various organelles, in many cases through unknown mechanisms. Furthermore, there are several types of RNA granules, many of which are sites of mRNA degradation; these are frequently found associated with membrane-bound organelles such as endosomes and mitochondria. In this review we summarize recent findings that link organelle function and mRNA localization to mRNA decay. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Salubrinal enhances eIF2α phosphorylation and improves fertility in a mouse model of Classic Galactosemia

Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT^?/?) mouse mimics the patient condition. GalT^?/? ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT^?/? adults. Here, we further characterized galactosemic stress in GalT^?/? mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT^?/? ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.     

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.