The heat shock response has been extensively studied in a variety of systems and organisms, and generally involves the conserved and coordinated upregulation of heat shock proteins that act to alleviate the cellular stresses imposed by hyperthermic stress. Our current understanding of the cellular responses to subphysiological temperatures (hypothermia) is less extensive. This is somewhat surprising, because of their relevance in medicine for the storage of cells, organs, and tissues, and the treatment of brain damage; as well as in the biopharmaceutical sector, where reduced culture temperature can sometimes improve recombinant protein yields from mammalian cells cultured in vitro [1]. What is clear is that the general response to hypothermia appears to include the global attenuation of transcription and translation, whereas a small group of proteins, termed the cold shock proteins, are selectively induced [2]. However, unlike their heat shock counterparts, these cold shock proteins do not appear to be particularly well conserved between prokaryotic and eukaryotic systems, and their functions, such as have been defined, have to date been described in terms of their RNA rather than their protein biology. Exposure to subphysiological temperature is also known to generally lead to changes in the lipid make-up of membranes, resulting in increased membrane rigidity, Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27-32°C) but became increasingly disrupted at low temperatures (4-10°C). The degradation rates of all mRNAs and proteins examined were much reduced at 27°C, and overall protein synthesis rates were gradually reduced with temperature down to 20°C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27°C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4°C), lipid-containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery.Abbreviations CCT, chaperonin containing T-complex polypeptide1; Cirp, cold-inducible RNA-binding protein; ER, endoplasmic reticulum; HSF, heat shock factor; NEPHGE, non-equilibrium pH gradient gel electrophoresis;...

SummaryCooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.

Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

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The molecular chaperone activities of the only known chaperonin in the eukaryotic cytosol (cytosolic chaperonin containing T-complex polypeptide 1 (CCT)) appear to be relatively specialized; the main folding substrates in vivo and in vitro are identified as tubulins and actins. CCT is unique among chaperonins in the complexity of its hetero-oligomeric structure, containing eight different, although related, gene products. In addition to their known ability to bind to and promote correct folding of newly synthesized and denatured tubulins, we show here that CCT subunits ␣, ␥, , and also associated with in vitro assembled microtubules, i.e. behaved as microtubule-associated proteins. This nucleotide-dependent association between microtubules and CCT polypeptides (K d ϳ 0.1 M CCT subunit) did not appear to involve whole oligomeric chaperonin particles, but rather free CCT subunits. Removal of the tubulin COOH termini by subtilisin digestion caused all eight CCT subunits to associate with the microtubule polymer, thus highlighting the non-chaperonin nature of the selective CCT subunit association with normal microtubules.Molecular chaperones are a diverse group of proteins that assist the correct folding and intracellular targeting of newly synthesized polypeptides (1) and can modulate the oligomerization and polymerization of folded native proteins (e.g. Ref.2). The chaperonins are a family of molecular chaperones that are characterized by their oligomeric structure, namely a double torus of ϳ60 kDa subunits (3) enclosing a central cavity within which the folding substrate may be sequestered (4 -6). Chaperonin-assisted protein folding proceeds by ATP-driven, alternating cycles of substrate binding and release, ultimately resulting in a native, or near-native, protein that is no longer recognized by the chaperonin (7,8). The cytosolic chaperonin containing T-complex polypeptide 1 (CCT) 1 is the only known chaperonin in the cytosol of eukaryotes (9, 10). The eightmembered rings of the CCT double torus consist usually of eight distinct but related (ϳ30% identity) gene products, CCT␣, -␤, -␥, -␦, -⑀, -, -, and -(11). In yeast, these eight subunits are encoded by essential genes, and mutations in individual subunits lead to defects in the functioning of the cytoskeleton, most commonly manifested as arrest in mitosis (reviewed in Ref. 12). There is both in vivo (10) and in vitro (13-15) evidence that major substrates of the cytosolic chaperonin are tubulins and actins. In addition to assisting folding of newly synthesized tubulins and actins, the CCT␣ subunit appears to be a component of the centrosome and essential for nucleated microtubule assembly from this organelle (16). That this process can take place in a permeabilized cell system, in the absence of protein synthesis, suggests that CCT␣ at least may also be involved in facilitating the polymerization of fully folded tubulins; which, if any, other CCT subunits are required remains to be determined. Indeed, whether CCT subunits always and only exist in cells as c...