Tailless Complex Polypeptide Research Articles

Folding for the Immune Synapse: CCT Chaperonin and the Cytoskeleton.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell’s intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.

Sequestration of the Transcription Factor STAT3 by the Molecular Chaperone CCT: A Potential Mechanism for Modulation of STAT3 Phosphorylation

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is an essential molecular chaperone required for the folding of the abundant proteins actin and tubulin. The CCT oligomer also folds a range of other proteins and participates in non-folding activities such as providing assembly support for complexes of the von Hippel Lindau tumor suppressor protein and elongins. Here we show that the oncogenic transcription factor STAT3 binds to the CCT oligomer, but does not display the early binding upon translation in rabbit reticulocyte lysate typical of an obligate CCT folding substrate. Consistent with this, depletion of each of the CCT subunits by siRNA targeting indicates that loss of CCT oligomer does not suppress the activation steps of STAT3 upon stimulation with IL-6: phosphorylation, dimerisation and nuclear translocation. Furthermore, the transcriptional activity of STAT3 is not negatively affected by reduction in CCT levels. Instead, loss of CCT oligomer in MCF7 cells leads to an enhancement of STAT3 phosphorylation at Tyr705, implicating a role for the CCT oligomer in the sequestration of non-phosphorylated STAT3. Thus, as CCT is dynamic oligomer, the assembly state and also abundance of CCT oligomer may provide a means to modulate STAT3 phosphorylation.

MTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

Effects of Stress Stimulation on Protein Folding/Degradation Pathway Related Proteins in Different Levels of Exhaustive Exercise

The pathways related to cell protein folding and degradation need to be elucidated because of training was set. The results showed that chaperonin containing tailless complex polypeptide the large number of proteins involved, the complex relationship between proteins and the lack of research on the effects of movement and cell protein folding and degradation. In order to analyze the effect of stress stimulation on protein folding/degradation pathway related proteins under different levels of exhaustive exercise, C57 Black 6 male mice of 4 w old were selected from the experimental animal center of the Academy of Military Medical Sciences and different intensity of treadmill exercise 1 beta, protein subunit alpha type-1 and protein subunit beta type-7 protein in insulin resistance mice bone increased significantly and insulin resistance improved significantly after 6 w of aerobic treadmill exercise. High intensity endurance exercise increased the expression of collagen volume fraction and type I collagen in right atrium and right ventricle (p<0.01) and the longer exercise time, the greater the increased range, and had no significant effect on the expression of collagen volume fraction and type I collagen in left ventricle. Although moderate intensity exercise promoted the expression of collagen volume fraction and collagen I in myocardium, there was no statistical significance.

Dlec1 is required for spermatogenesis and male fertility in mice

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1−/− mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet–Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

Correlated fluorescence microscopy and multi-ion beam secondary ion mass spectrometry imaging reveals phosphatidylethanolamine increases in the membrane of cancer cells over-expressing the molecular chaperone subunit CCT\u03b4

Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C60+ and (CO2)6k+ ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCTδ, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation.Graphical abstract

The Molecular Chaperone CCT/TRiC: An Essential Component of Proteostasis and a Potential Modulator of Protein Aggregation.

Chaperonin containing tailless complex polypeptide 1 (CCT) or tailless complex polypeptide 1 ring complex (TRiC) is an essential eukaryotic molecular chaperone. It is a multi-subunit oligomer of two rings of eight individual protein subunits. When assembled, each of the eight CCT subunits occupies a specific position within each chaperonin ring. Thus a geometrically defined binding interface is formed from the divergent sequences within the CCT subunit substrate binding domains. CCT is required for the folding of the abundant cytoskeletal proteins actin and tubulin, which in turn form assemblies of microfilaments and microtubules. CCT is also involved in the folding of some additional protein substrates and some CCT subunits have been shown to have functions when monomeric. Since observations were made in worms over a decade ago using an RNAi screen, which connected CCT subunits to the aggregation of polyglutamine tracts, a role for CCT as a potential modulator of protein aggregation has started to emerge. Here there will be a focus on how mechanistically CCT may be able to achieve this and if this potential function of CCT provides any insights and directions for developing future treatments for protein aggregation driven neurodegenerative diseases generally, many of which are associated with aging.

Overexpressing CCT6A Contributes To Cancer Cell Growth By Affecting The G1-To-S Phase Transition And Predicts A Negative Prognosis In Hepatocellular Carcinoma.

PurposeTo determine the oncogenic role of the sixth subunit of chaperonin-containing tailless complex polypeptide 1 (CCT6A) in hepatocellular carcinoma (HCC) and address the correlation of CCT6A with clinicopathological characteristics and survival. Additionally, this study aimed to explore the effect of CCT6A on HCC cells and the underlying mechanisms.MethodsWe searched for levels of CCT6A expression in the Oncomine database and GEPIA database, which was then validated by analyzing cancer and adjacent non-cancerous tissues of HCC patients using quantitative PCR, Western blot, and immunohistochemistry assays. The relationship between CCT6A expression and survival was analyzed from the GEPIA database and confirmed by immunohistochemistry assays of 133 HCC tissue sections. In addition, the effect of depleting CCT6A on cell proliferation was assessed by CCK-8 and colony formation assays. Cell cycle analysis, immunofluorescence assays, GSEA analysis, and cyclin D expression analyzed by Western blot were used to explore the possible underlying mechanism how dysregulated CCT6A affect the proliferation of HCC.ResultsBoth mRNA and protein levels of CCT6A were increased in HCC tissues. Higher CCT6A expression was significantly associated with reduced overall survival (P = 0.023). CCT6A depletion inhibited cell proliferation and downregulated cyclin D, hindering the G1-to-S phase arrest.ConclusionCCT6A may contribute to HCC cell proliferation by accelerating the G1-to-S transition, as it maintains the expression of cyclin D. CCT6A could be considered an oncogene of HCC and could be used as a prognostic biomarker for HCC.

Cdc20 and molecular chaperone CCT2 and CCT5 are required for the Muscovy duck reovirus p10.8-induced cell cycle arrest and apoptosis

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.

Murine cytomegalovirus M72 promotes acute virus replication in vivo and is a substrate of the TRiC/CCT complex

Betaherpesvirus dUTPase homologs are core herpesvirus proteins, but little is known about their role during infection. Human cytomegalovirus (HCMV) UL72 and murine cytomegalovirus (MCMV) M72 have been designated dUTPase homologs, and previous studies indicate UL72 is dispensable for replication and enzymatically inactive. Here, we report the initial characterization of MCMV M72. M72 does not possess dUTPase activity, and is expressed as a leaky-late gene product with multiple protein isoforms. Importantly, M72 augments MCMV replication in vitro and during the early stage of acute infection in vivo. We identify and confirm interaction of M72 with the eukaryotic chaperonin tailless complex protein -1 (TCP-1) ring complex (TRiC) or chaperonin containing tailless complex polypeptide 1 (CCT). Accumulating biochemical evidence indicates M72 forms homo-oligomers and is a substrate of TRiC/CCT. Taken together, we provide the first evidence of M72's contribution to viral pathogenesis, and identify a novel interaction with the TRiC/CCT complex.

Interactions between monomeric CCTδ and p150Glued: A novel function for CCTδ at the cell periphery distinct from the protein folding activity of the molecular chaperone CCT

Chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone consisting of eight distinct protein subunits, that when oligomeric is essential for the folding of newly synthesized tubulin and actin. In addition to folding, CCT activity includes functions of individual subunits in their monomeric form. For example, when CCTδ monomer levels are increased in cultured mammalian cells, numerous cell surface protrusions are formed from retraction fibres, indicating that an underlying function for the CCTδ monomer exists. Here, using a yeast two-hybrid screen we identify the dynactin complex component p150Glued as a binding partner for CCTδ and show by siRNA depletion that this interaction is required for the formation of CCTδ-induced cell surface protrusions. Intact microtubules are necessary for the formation of the protrusions, consistent with microtubule minus end transport driving the retraction fibre formation and depletion of either p150Glued or the dynactin complex-associated transmembrane protein dynAP prevents the previously observed localization of GFP-CCTδ to the plasma membrane. Wound healing assays reveal that CCTδ monomer levels influence directional cell migration and together our observations demonstrate that in addition to the folding activity of CCT in its oligomer form, a monomeric subunit is associated with events that involve the assembled cytoskeleton.

Genetic expansion of chaperonin-containing TCP-1 (CCT/TRiC) complex subunits yields testis-specific isoforms required for spermatogenesis in planarian flatworms.

Chaperonin-containing Tail-less complex polypeptide 1 (CCT) is a highly conserved, hetero-oligomeric complex that ensures proper folding of actin, tubulin, and regulators of mitosis. Eight subunits (CCT1-8) make up this complex, and every subunit has a homolog expressed in the testes and somatic tissue of the planarian flatworm Schmidtea mediterranea. Gene duplications of four subunits in the genomes of S. mediterranea and other planarian flatworms created paralogs to CCT1, CCT3, CCT4, and CCT8 that are expressed exclusively in the testes. Functional analyses revealed that each CCT subunit expressed in the S. mediterranea soma is essential for homeostatic integrity and survival, whereas sperm elongation defects were observed upon knockdown of each individual testis-specific paralog (Smed-cct1B; Smed-cct3B; Smed-cct4A; and Smed-cct8B), regardless of potential redundancy with paralogs expressed in both testes and soma (Smed-cct1A; Smed-cct3A; Smed-cct4B; and Smed-cct8A). Yet, no detriment was observed in the number of adult somatic stem cells (neoblasts) that maintain differentiated tissue in planarians. Thus, expression of all eight CCT subunits is required to execute the essential functions of the CCT complex. Furthermore, expression of the somatic paralogs in planarian testes is not sufficient to complete spermatogenesis when testis-specific paralogs are knocked down, suggesting that the evolution of chaperonin subunits may drive changes in the development of sperm structure and that correct CCT subunit stoichiometry is crucial for spermiogenesis.

Elevated chaperone proteins are a feature of winter freeze avoidance by larvae of the goldenrod gall moth, Epiblema scudderiana

Winter survival for many insect species includes a need to maintain metabolic homeostasis and structural/functional integrity of macromolecules not only over a wide range of cold temperatures but also in response to rapid temperature change. Chaperones are well-known to protect/stabilize protein structure with regard to heat stress but less is known about their potential involvement in long-term protection of the proteome at subzero temperatures. The present study assessed the participation of chaperone proteins in the cold hardiness of larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae), monitoring changes in nine proteins over the winter months as well as their responses to laboratory cold acclimation or anoxia exposure. Four heat shock proteins (HSPs: Hsp110, Hsp70, Hsp60, Hsp40), three glucose-regulated proteins (GRPs: Grp78, Grp 94, Grp170) and the tailless complex polypeptide 1 (TCP-1) as well as the heat shock transcription factor (HSF1) were investigated. In general, all were significantly elevated in larvae collected from an outdoor site between October and March, as compared with September values, and chaperone levels were reduced again in April. The October to March interval also includes the period of diapause followed by cold quiescence in the species. Relative expression of Hsp70, Hsp60 and Hsp40 rose by 2–3-fold, GRPs increased 1.5–3-fold, and levels of active (hyperphosphorylated) HSF1 increased by 4–4.8-fold over the midwinter months. Chilling from 15°C to 4°C in the laboratory upregulated Grp78 protein content that remained high as temperature was further reduced to −4°C and then −20°C whereas Hsp110, Hsp70 and HSF1 levels increased when larvae were exposed to −4°C and −20°C. Grp170 (also known as oxygen-regulated protein 150) was the only chaperone that increased significantly in the larvae in response to anoxia exposure at 4°C. The data also indicated that multiple subcellular compartments received enhanced protection for their proteome since upregulation of chaperones included proteins known to occur in cytosolic (Hsp40, Hsp70), mitochondrial (Hsp60) and endoplasmic reticulum (Grp170) locations. Overall, the data indicate that chaperones have a significant role to play in the winter cold hardiness of E. scudderiana and identify declining temperatures (and perhaps also oxygen restriction) as potential modulators of chaperone production. The data add support to a relatively understudied area of insect cold hardiness – the long-term protection and stabilization of the proteome over the winter months.

Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.

The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h

eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.

Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding

Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT-folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 bound the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Molecular functions of chaperonin gene, containing tailless complex polypeptide 1 from Macrobrachium rosenbergii

Chaperonin (MrChap) was identified from a constructed transcriptome dataset of freshwater prawn Macrobrachium rosenbergii. The MrChap peptide contains a long chaperone super family domain between 11 and 525. Three chaperone tailless complex polypeptide (TCP-1) signatures are present in the MrChap peptide sequence at 36–48, 57–73 and 85–93. The gene expressions of MrChap in both healthy M. rosenbergii and those infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV) were examined using qRT-PCR. To understand its biological activity, the recombinant MrChap gene was constructed and expressed in Escherichia coli BL21 (DE3). The results of ATPase assay showed that the recombinant MrChap protein exhibited apparent ATPase activity. Chaperone activity assay showed that the recombinant MrChap protein is an active chaperone. These results suggest that MrChap is potentially involved in the immune responses against viral infection in M. rosenbergii. These findings indicate that the recombinant MrChap protein may be used in immunotherapeutic approaches.

Chaperone proteins and winter survival by a freeze tolerant insect

The role of chaperone proteins in the winter survival of insects was evaluated in freeze tolerant gall fly larvae, Eurosta solidaginis. Levels of four heat shock proteins (Hsp110, Hsp70, Hsp60, Hsp40), two glucose-regulated proteins (Grp75, Grp78) and three others (tailless complex polypeptide 1 [TCP-1], αA-crystallin, αB-crystallin) were tracked in outdoor larvae from September to April and, in addition, laboratory experiments assessed chilling, freezing, and anoxia effects on these proteins. Gall fly larvae showed consistent elevation of Hsp110, Hsp70, Hsp40, Grp78 and αB-crystallin over the late autumn and winter months, generally 1.5–2.0-fold higher than September values. This suggests that these proteins contribute to cell preservation over the winter months via protection and stabilization of macromolecules. By contrast, levels of the mitochondrial Hsp60 fell to just 40% of September values by midwinter, paralleling the responses by numerous mitochondrial enzymes and consistent with a reduction in total mitochondria numbers over the winter. None of the proteins were altered when 15 °C acclimated larvae were chilled to 3 °C for 24 h but Hsp70, Hsp40 and Grp75 increased during freezing at −16 °C for 24 h whereas others (Hsp110, TCP-1 and both crystallins) increased significantly after larvae thawed at 3 °C. Anoxia exposure (24 h under N2 gas at 15 °C) elevated levels of Hsp70, Grp78 and the two crystallins. Levels of active hyperphosphorylated heat shock transcription factor (HSF1) were also analyzed, giving an indication of the state of hsp gene transcription in the larvae. HSF1 was high in September and October but fell to less than 40% of September values in midwinter consistent with suppression of gene transcription in diapause larvae. HSF1 levels responded positively to freezing and increased robustly by 4.9-fold under anoxia. Overall, the data provide strong evidence for the importance of protein chaperones as a mechanism of cell preservation in freeze tolerant insects.

Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.