Chaperonin-containing T-complex Research Articles

TCP1 expression alters the ferroptosis sensitivity of diffuse large B-cell lymphoma subtypes by stabilising ACSL4 and influences patient prognosis

Diffuse large B-cell lymphoma (DLBCL), an invasive lymphoma with substantial heterogeneity, can be mainly categorised into germinal centre B-cell-like (GCB) and non-GCB subtypes. DLBCL cells are highly susceptible to ferroptosis, which offers an effective avenue for treating recurrent and refractory DLBCL. Moreover, various heat shock proteins are involved in regulating the sensitivity of tumour cells to ferroptosis. Among these proteins, tailless complex polypeptide 1 (TCP1), a subunit of chaperonin-containing T-complex protein-1 (CCT), plays a role in tumour proliferation and survival. Therefore, we explored the role of TCP1 in different DLBCL subtypes, the sensitivity of GCB and non-GCB subtypes to the ferroptosis inducer RAS-selective lethal small molecule 3 (RSL3), and the underlying molecular mechanism. In GCB cells, TCP1 promoted RSL3-induced ferroptosis. Notably, TCP1 could bind with acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme regulating lipid composition and facilitating ferroptosis, to reduce its ubiquitination and degradation. This interaction activated the ACSL4/LPCAT3 signalling pathway and promoted ferroptosis in the GCB subtype. However, in the non-GCB subtype, TCP1 did not act as a positive regulator but served as a predictor of an unfavourable prognosis in patients with non-GCB. In conclusion, our results suggest that in DLBCL, high TCP1 expression enhances the sensitivity of GCB tumour cells to ferroptosis and serves as a marker of poor prognosis in patients with non-GCB DLBCL.

Complementary DNA Cloning and Tissue Expression Profile of Chaperonin-Containing T-Complex Polypeptide 1-Beta in Pinctada maxima Exposed to Cold Stress

Complementary DNA Cloning and Tissue Expression Profile of Chaperonin-Containing T-Complex Polypeptide 1-Beta in Pinctada maxima Exposed to Cold Stress

Relative Abundance of Spermadhesin-1 in the Seminal Plasma of Young Nellore Bulls Is in Agreement with Reproductive Parameters.

This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection.

Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus.

Suid herpesvirus 1 (SuHV-1), known as pseudorabies virus (PRV), is one of the most devastating swine pathogens in China, particularly the sudden occurrence of PRV variants in 2011. The higher pathogenicity and cross-species transmission potential of the newly emerged variants caused not only colossal economic losses, but also threatened public health. To uncover the underlying pathogenesis of PRV variants, Tandem Mass Tag (TMT)-based proteomic analysis was performed to quantitatively screen the differentially expressed cellular proteins in PRV-infected Vero cells. A total of 7072 proteins were identified and 960 proteins were significantly regulated: specifically 89 upregulated and 871 downregulated. To make it more credible, the expression of XRCC5 and XRCC6 was verified by western blot and RT-qPCR, and the results dovetailed with the proteomic data. The differentially expressed proteins were involved in various biological processes and signaling pathways, such as chaperonin-containing T-complex, NIK/NF-κB signaling pathway, DNA damage response, and negative regulation of G2/M transition of mitotic cell cycle. Taken together, our data holistically outline the interactions between PRV and host cells, and our results may shed light on the pathogenesis of PRV variants and provide clues for pseudorabies prevention.

Suppression of CCT3 Inhibits Tumor Progression by Impairing ATP Production and Cytoplasmic Translation in Lung Adenocarcinoma

Heat shock proteins are highly expressed in various cancers and exert critical functions in tumor progression. However, their expression patterns and functions in lung adenocarcinoma (LUAD) remain largely unknown. We identified that chaperonin-containing T-complex protein-1 subunit 3 (CCT3) was highly expressed in LUAD cells and was positively correlated with LUAD malignancy in the clinical samples. Animal studies showed that silencing CCT3 dramatically inhibited tumor growth and metastasis of LUAD. Proliferation and migration were markedly suppressed in CCT3-deficient LUAD cells. Moreover, the knockdown of CCT3 promoted apoptosis and cell cycle arrest. Mechanistically, the function of glycolysis was significantly inhibited and the total intracellular ATP levels were reduced by at least 25% in CCT3-deficient cells. In addition, the knockdown of CCT3 decreased the protein translation and led to a significant reduction in eukaryotic translation initiation factor 3 (EIF3G) protein, which was identified as a protein that interacts with CCT3. Impaired protein synthesis and cell growth in EIF3G-deficient cells were consistent with those caused by CCT3 knockdown in LUAD cells. Taken together, our study demonstrated in multiple ways that CCT3 is a critical factor for supporting growth and metastasis of LUAD, and for the first time, its roles in maintaining intracellular ATP levels and cytoplasmic translation are reported. Our novel findings provide a potential therapeutic target for lung adenocarcinoma.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact.

Cdna Cloning and Expression Analysis of Chaperonin Containing T-Complex Polypeptide 1-Alpha in the Pearl Oyster Pinctada maxima Exposed to Cold Stress

The complete cDNA sequence of chaperonin-containing T-complex polypeptide 1-alpha (CCT-alpha) gene of the pearl oyster Pinctada maxima was determined using rapid amplification of cDNA ends technique and characterized with bioinformatic analysis. Its expression profile in different organs and under cold stress was characterized using real-time quantitative polymerase chain reaction. Results revealed that the complete cDNA sequence of P. maxima CCT-alpha has 1,803 base pairs (bp), including a 1,608-bp open reading framework that codes 535 amino acids. The protein has three conservative functional domains: equatorial, apical, and intermediate domains. The equatorial domain is 20 amino acids shorter than that of other species, and contains the three signature sequences of CCT family protein. The CCT-alpha of P. maxima has nine conservative ATP-binding sites and six protein-binding sites, and has high homology with Aplysia californica (80.7%), Delia antiqua (77.4%), Danio rerio (77.1%), Xenopus laevis (76.7%), Gallus gallus (76.4%), and Homo sapiens (75.4%). The CCT-alpha shows tissue-specific expression profile in P. maxima, with expression levels higher in gill, mantle, and adductor muscle than in hepatopancreas and heart. Cold stress markedly increased mRNA contents of CCT-alpha in the organs of P. maxima, but different organs showed distinct cold stress response pattern. Chaperonin-containing T-complex polypeptide 1-alpha transcription in adductor muscle, hepatopancreas, and heart increased at 2 h, and reached peak values at 6 h, maintained high levels at 12 h, and significantly decreased at 24 h. The product in gill and mantle decreased at 2 h, began to increase at 6 h, and showed a large increase at 12 h, but reduced to below the initial level at 24 h.

CCT4 suppression inhibits tumor growth in hepatocellular carcinoma by interacting with Cdc20.

Background:The chaperonin containing t-complex (CCT) proteins play an important role in cell cycle-related protein degradation in yeast and mammals. The role of the chaperonin containing t-complex 4 (CCT4), one subtype of CCT proteins, in the progress of hepatocellular carcinoma (HCC) was not fully elucidated. Here, we aimed to explore the mechanisms of CCT4 in HCC.Methods:In this study, we used the UALCAN platform to analyze the relationship between CCT4 and HCC, and the association of CCT4 with the overall survival (OS) of HCC patients was also analyzed. CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot (WB) assay. Lentivirus vector was used to knock down the CCT4 expression, and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines. Cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to detect the cell proliferation, and flow cytometry (FCM) was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells. Co-immunoprecipitation (co-IP) assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC. Data were calculated from at least three replicate experiments and expressed as mean ± standard deviation. Student's t test, paired t test, and Kaplan–Meier analysis were used to compare across different groups.Results:We found CCT4 was upregulated in HCC tissues compared with normal tissues, and its high expression was associated with poor prognosis (P < 0.001). CCT4 was significantly increased in HCC tumor tissues compared with normal tissues (0.98 ± 0.12 vs. 0.23 ± 0.05, t = 7.73, P < 0.001). After being transfected with CCT4 short-hairpin RNA (shRNA), CCT4 was decreased in mRNA level and protein level in both Huh7 (mRNA level: 0.41 ± 0.07 vs. 1.01 ± 0.11, t = 8.09, P = 0.001; protein level: 0.61 ± 0.03 vs. 0.93 ± 0.07, t = 7.19, P = 0.002) and Hep3b cells (mRNA level: 0.55 ± 0.11 vs. 1.04 ± 0.15, t = 4.51, P = 0.011; protein level: 0.64 ± 0.10 vs. 0.95 ± 0.08, t = 4.32, P = 0.012). CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7 (OD value of 3 days: 0.60 ± 0.14 vs. 0.97 ± 0.16, t = 3.13, P = 0.036; OD value of 4 days: 1.03 ± 0.07 vs. 1.50 ± 0.12, t = 5.97, P = 0.004) and Hep3b (OD value of 3 days: 0.69 ± 0.14 vs. 1.10 ± 0.11, t = 3.91, P = 0.017; OD value of 4 days: 1.12 ± 0.12 vs. 1.48 ± 0.13, t = 3.55, P = 0.024) cells. EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7 (EdU positive rate: [31.25 ± 3.41]% vs. [58.72 ± 3.78]%, t = 9.34, P = 0.001) and Hep3b cells (EdU positive rate: [44.13 ± 7.02]% vs. [61.79 ± 3.96]%, t = 3.79, P = 0.019). FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells (apoptosis rate of Huh7: [9.10 ± 0.80]% vs. [3.66 ± 0.64]%, t = −9.18, P = 0.001; apoptosis rate of Hep3b: [6.69 ± 0.72]% vs. [4.20 ± 0.86]%, t = −3.84, P = 0.018). We also found that CCT4 could regulate anaphase-promoting complex (APC)Cdc20 activity via interacting with Cdc20. Furthermore, CCT4 knockdown induced securin (0.65 ± 0.06 vs. 0.44 ± 0.05, t = −4.69, P = 0.009) and B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (Bim; 0.96 ± 0.06 vs. 0.61 ± 0.09, t = −5.65, P = 0.005) accumulation. The upregulation of securin inhibited cell growth by downregulating cyclin D1 (0.65 ± 0.05 vs. 1.04 ± 0.07, t = 8.12, P = 0.001), and the accumulation of Bim inhibited Bcl-2 (0.77 ± 0.04 vs. 0.87 ± 0.04, t = 3.00, P = 0.040) and activated caspase 9 (caspase 9: 0.77 ± 0.04 vs. 0.84 ± 0.05, t = 1.81, P = 0.145; cleaved caspase 9: 0.64 ± 0.06 vs. 0.16 ± 0.07, t = 1.81, P = 0.001), which led to elevated apoptosis.Conclusions:Overall, these results showed that CCT4 played an important role in HCC pathogenesis through, at least partly, interacting with Cdc20.

PSXIII-5 Effects of B vitamins and hydroxy trace minerals supplementation on hepatic metabolism of beef cattle at finishing phase in pasture

Abstract Vitamin B and trace minerals are crucial molecular signals involved in many biological pathways of energy metabolism and their bioavailability is compromised in high-producing ruminant animals. Despite the current knowledge of the effects of vitamin B complex and trace minerals on animal performance, their use as a rumen-protected form and its impact on liver metabolism in finishing beef cattle is poorly known. The present study aimed to assess the effects of ruminally protected B-vitamin blend (Trouw Nutrition, Italy) and hydroxy trace minerals (Cu and Zn; Micronutrients Inc., Indianapolis, IN) on hepatic proteome. A total of 20 non-castrated Nellore males with 353 ± 43 kg of initial BW were submitted in a high supplementation (1.7% of BW) in pasture and randomly assigned to one of the following treatments: CTRL – inorganic trace minerals without supplementation of vitamin B blend; SUP – supplementation of hydroxy trace minerals (Cu and Zn) and vitamin B blend (B5, B6, B7, B9, and B12). All animals were fed the same level of the experimental diet for 106 days and liver biopsy was performed at the end of the experimental period. We use shotgun proteomics combined with biological and network analyses of the protein differentially abundant between treatments, showing 37 proteins differentially abundant (P &amp;lt; 0.10) between treatment groups, where all proteins were up-regulated in the SUP treatment. These proteins were related to protein folding (P = 0.0456), oxidoreductase complex (P = 0.0155), mitochondrial respiratory chain complex IV (P = 0.0132), chaperonin-containing T-complex 2 (P = 0.0132), glutathione metabolism (P = 0.00026) and other linked to oxidative stress regulation. These results indicate that ruminal-protected vitamin B and hydroxy trace minerals supplementation during the finishing phase cause positive changes in the abundance of proteins related to oxidative metabolism and response to oxidative damage.

Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex

Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.

CCTδ colocalizes with actin and β-tubulin: Insight into its involvement in the cytoskeleton formation of the intracellular parasite Nosema bombycis

The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone protein that is widely present in eukaryotic cytoplasm and can assist in the folding of newly synthesized proteins. The CCT complex consists of eight completely different subunits, among which the δ subunit plays an extremely important role in the folding and assembly of cytoskeleton proteins as an individual or complex with other subunits. In this study, we identified the CCTδ in the microsporidian Nosema bombycis (NbCCTδ) for the first time. The NbCCTδ gene contains a complete ORF of 1497 bp in length that encodes a 498 amino acid polypeptide. NbCCTδ is expressed throughout the entire lifecycle of N. bombycis and rather higher in early stage of proliferation. Indirect immunofluorescence results showed that NbCCTδ was colocalized with actin and β-tubulin during the proliferative and sporogonic phases of N. bombycis. RNA interference down-regulated the expression of the NbCCTδ gene. These results imply that NbCCTδ may participate in cytoskeleton formation and proliferation of N. bombycis.

Proteomic Analysis of Liver from Finishing Beef Cattle Supplemented with a Rumen-Protected B-Vitamin Blend and Hydroxy Trace Minerals.

Simple SummaryGreater metabolic needs in high-producing beef cattle might lead to mineral and vitamin deficiency. Previous studies have shown the benefits of B-vitamin and trace mineral supplementation in animal performance of ruminants; however, little is known about the effects of supplementing finishing beef cattle with rumen-protected forms on the liver metabolism. Therefore, the aim of the present study was to determinate the impact of rumen-protected B-vitamin blend and hydroxy trace mineral supplementation on the hepatic proteome of finishing steers. This study reports the first evidence indicating that the supplementation of these micronutrients induces protein changes concerning oxidative metabolism and responses to oxidative stress in the liver tissue.Vitamin B and trace minerals are crucial molecular signals involved in many biological pathways; however, their bioavailability is compromised in high-producing ruminant animals. So far, studies have mainly focused on the effects of these micronutrients on animal performance, but their use in a rumen-protected form and their impact on liver metabolism in finishing beef cattle is poorly known. We used a shotgun proteomic approach combined with biological network analyses to assess the effects of a rumen-protected B-vitamin blend, as well as those of hydroxy trace minerals, on the hepatic proteome. A total of 20 non-castrated Nellore males with 353 ± 43 kg of initial body weight were randomly assigned to one of the following treatments: CTRL—inorganic trace minerals without supplementation of a protected vitamin B blend, or SUP—supplementation of hydroxy trace minerals and a protected vitamin B blend. All animals were fed the same amount of the experimental diet for 106 days, and liver biopsies were performed at the end of the experimental period. Supplemented animals showed 37 up-regulated proteins (p < 0.10), and the enrichment analysis revealed that these proteins were involved in protein folding (p = 0.04), mitochondrial respiratory chain complex I (p = 0.01) and IV (p = 0.01), chaperonin-containing T-complex 2 (p = 0.01), glutathione metabolism (p < 0.01), and other aspects linked to oxidative-stress responses. These results indicate that rumen-protected vitamin B and hydroxy trace mineral supplementation during the finishing phase alters the abundance of proteins associated with the electron transport chain and other oxidation–reduction pathways, boosting the production of reactive oxygen species, which appear to modulate proteins linked to oxidative-damage responses to maintain cellular homeostasis.

Sperm proteomic changes associated with early embryo quality after ICSI

Do alterations of human sperm protein profile affect embryo quality? Sperm proteins from 27 infertile couples undergoing intracytoplasmic sperm injection (ICSI) were extracted and digested. The resulting peptides were labelled using tandem mass tags, separated by two-dimensional liquid chromatography, and identified and quantified using tandem mass spectrometry. Subsequently, sperm protein and peptide abundance were statistically analysed for correlation with ICSI-derived embryo quality in the subset of idiopathic infertile couples. Detected correlations were further assessed in the subset of infertile patients with a known factor. The abundance of 18 individual sperm proteins was found to correlate with embryo quality after ICSI. Of note, a high percentage of poor-quality ICSI-derived embryos was associated with alterations in several components of the eight-membered chaperonin-containing T-complex, which plays an important role in the folding of many essential proteins. Additionally, the abundance of sperm proteins with known functions in embryogenesis, such as RUBVL1, also correlated with early embryo quality (r = -0.547; P = 0.028). Some of the correlations found in this study were validated using either proteomic data from infertile patients with a known factor or data from similar published studies. Analysis at the peptide level revealed the association of some correlations with specific post-translational modifications or isoforms. Our results support the hypothesis that the sperm proteome plays a role in early embryogenesis. Moreover, several sperm proteins have emerged as potential biomarkers that could predict the outcome of in-vitro assisted reproductive technologies, leading to the possibility of improved diagnosis of couples with idiopathic infertility.

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC.

The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT), plays an important role in cytosolic proteostasis. It has been estimated that as much as 10% of cytosolic proteins interact with CCT during their folding process. CCT is composed of 8 different paralogous subunits. Due to its complicated structure, molecular and biochemical investigations of CCT have been difficult. In this study, we constructed an expression system for CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT), by using E. coli as a host. As expected, we obtained recombinant CtCCT with a relatively high yield, and it exhibited fairly high thermal stability. We showed the advantages of the overproduction system by characterizing CtCCT variants containing ATPase-deficient subunits. For diffracted X-ray tracking experiment, we removed all surface exposed cysteine residues, and added cysteine residues at the tip of helical protrusions of selected two subunits. Gold nanocrystals were attached onto CtCCTs via gold-thiol bonds and applied for the analysis by diffracted X-ray tracking. Irrespective of the locations of cysteines, it was shown that ATP binding induces tilting motion followed by rotational motion in the CtCCT molecule, like the archaeal group II chaperonins. When gold nanocrystals were attached onto two subunits in the high ATPase activity hemisphere, the CtCCT complex exhibited a fairly rapid response to the motion. In contrast, the response of CtCCT, which had gold nanocrystals attached to the low-activity hemisphere, was slow. These results clearly support the possibility that ATP-dependent conformational change starts with the high-affinity hemisphere and progresses to the low-affinity hemisphere.

Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+ concentrations and (ii) by measuring their sensitivity to the mutation Gly345→Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+ concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that “conformational selection” by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism.

Proteomic and Genomic Analyses of the Rvb1 and Rvb2 Interaction Network upon Deletion of R2TP Complex Components

The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.

Abstract P5-04-04: Cutting the ties that bind: Targeting chaperonin-containing T-complex (CCT) for therapeutic intervention in the treatment of advanced stage breast cancer

Abstract Metastatic disease is a principal cause of death from breast cancer. This is due in part to the development of resistance to current therapeutics and the often debilitating side effects that impair quality of life. The challenge is to therapeutically target an essential physiological function of cancer cells not found in normal, non-transformed cells. To this end, we discovered CT20p, a therapeutic peptide that causes cancer-specific death in human breast tumor cells and tumor regression in xenograft models of breast cancer. Using a proteomics approach, we found that CT20p directly binds to multiple subunits of a type II chaperonin called chaperonin containing T-complex or CCT. CCT forms a large macromolecular complex composed of 8 subunits (CCTα, CCTβ, CCTγ, CCTδ, CCTϵ, CCTζ, CCTη, CCTω). Each subunit contains an ATP binding site as well as substrate binding sites. Inhibition of CCT by CT20p depletes the pool of native actin and tubulin (obligate clients of CCT), impairing the polymerization of cytoskeletal elements needed to support cell adhesion and motility. As a result cancer cells lose the ability to migrate and die from loss of substrate survival signals. We found that expression levels of CCT varied among different triple negative breast cancer (TNBC) cell lines, with the highest expression occurring in those of the mesenchymal stem-like (MSL) subtype with metastatic potential. Lowest levels of CCT were found in normal breast epithelial cells. Sensitivity to killing by CT20p thus correlated with levels of CCT, with cancer cells expressing high amounts of CCT being the most susceptible. Using tissue microarrays (TMAs) of breast cancer progression, we developed an immunohistochemistry staining procedure for CCTβ. Results were interpreted on a scale of 1 to 4 (with 4 being the strongest staining). CCTβ expression was statistically higher in invasive ductal carcinoma (IDC) as compared to normal and cancer adjacent tissue (CAT) (p&amp;lt;0.0001). Within the types of IDC, CCTβ was highest in tumors that exceeded 5 cm across (T3), grew in chest wall or skin and in inflammatory breast cancer (T4) (p&amp;lt;0.05). Examining CCTβ levels in different molecular types showed little correlation with estrogen receptor (ER) positivity but strong correlation with ER and progesterone receptor (PR) positivity or PR alone (p&amp;gt;0.001). Statistical correlations were also observed with Her2 positivity (p&amp;gt;0.05). However, no statistically significant correlations were observed with TNBC tissues, with CCTβ staining ranging from the strongest staining (4) to lowest (1). These results are similar to that observed with the TNBC cell lines and indicate that CCT expression may reflect the heterogeneity of TNBC. These results suggest that CCT is a promising target for therapeutic intervention due to its increased expression in advanced stage breast cancer, independence of molecular identity and dependence by cancer cells to support essential cytoskeletal changes associated with the metastatic stem-like phenotype. Citation Format: Khaled AS, Vishnubhotla P, Khaled AR, Bassiouni R. Cutting the ties that bind: Targeting chaperonin-containing T-complex (CCT) for therapeutic intervention in the treatment of advanced stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-04.

Abstract P4-04-12: Enhancing the immunogenicity of breast cancer cells to stimulate innate immunity and augment the effects of cellular immunotherapy

Abstract A promising strategy for treating metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system and can be used in combination with checkpoint blockade or adoptive cell transfer to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to stimulate a tumor-specific immune response. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) and that could sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of a murine xenograft model with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of the "eat me" signal, calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT), which is essential for the folding of actin and tubulin and other critical proteins into their native forms. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that generate the danger signals associate with ICD. To this end, we observed that cancer cells treated with CT20p displayed alterations in PERK, a key mediator of the unfolded protein response (UPR) and translocation of Crt. As a result, CT20p-treated cancer cells were more readily phagocytosed. More importantly, NK cells more effectively killed cancer cells pre-treated with CT20p. This suggests that CT20p may stimulate NK cell cytotoxicity by inducing activating signals on cancer cells, which would greatly augment the curative effects of adoptive transfer of NK cells. In our experiments normal breast epithelial cells, macrophages or NK cells, themselves, were unaffected by CT20p treatment due reduced CCT. These studies indicate that a single molecule, CT20p, can do both the killing of cancer cells and activation of immune cells and can be combined with cellular immunotherapy (e.g. expanded NK cells) to provide a more complete protection from breast cancer recurrence and metastasis. Citation Format: Khaled AR, Limaye A, Bassiouni R, Showalter A, Oyer J, Pandey V, Igarashi R, Altomare DA, Copik AJ. Enhancing the immunogenicity of breast cancer cells to stimulate innate immunity and augment the effects of cellular immunotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-12.

Abstract P5-03-02: The dynamic duo: A breast cancer-targeting nanoparticle loaded with a cytotoxic peptide as a treatment for metastatic disease

Abstract Metastatic breast cancer is a uniformly fatal disease with a 5-year survival rate of 15 percent. To date there are no effective approaches for targeted therapy. To develop a treatment for metastatic cancer, our group discovered a novel cytotoxic peptide, CT20p, and developed a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. CT20p was derived from Bax, a member of the Bcl-2 family. Unlike the parent protein, CT20p does not cause apoptosis and its cytotoxicity is independent of caspases and Bcl-2 overexpression. Rather, the intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT), which is required for the folding of actin and tubulin into their native forms. Inhibition of CCT activity by CT20p, indicated by decreased F-actin and tubulin, impaired the polymerization of microfilaments and microtubules, causing loss of cell migration and adhesion that promoted breast cancer cell death. In contrast, normal, non-transformed cells were resistant to the cytotoxicity of CT20p. On its own, CT20p is not membrane-permeable. To deliver the peptide to cells, we used nanoparticles formed with a novel aliphatic hyperbranched polyester polymer (HBPE-NPs). The surface of HBPE-NPs retains carboxylic acid groups for labeling of targeting ligands to enable accumulation in tumors. To concentrate on breast cancer, we functionalized the HBPE-NPs with either folate (FOL) or glutamate (GLU), which target the folate receptor (FR) or the metabotropic glutamate receptor (GRM-1) respectively. FR and GRM-2 are essential metabolic components that are highly expressed in solid tumors like breast cancer. In vitro targeting studies using triple negative breast cancer cell (TNBC) lines established that folate FOL or GLU-HBPE-NPs loaded with fluorescent dyes were readily up taken at high efficiency by TNBC cells. HBPE-NPs also contain unique hydrophobic cavities especially suited for encapsulating CT20p. We found that once the CT20p-HBPE-NPs were taken up by cancer cells, the peptide was released inside cells under acidic conditions (e.g. endosomes) and directly interacted with its intracellular target, CCT. Studies using primary cells derived from human breast tumors confirmed the targeted uptake of HBPE-NPs as well as demonstrated the cancer-specific cytotoxicity of CT20p. We treated a murine TNBC xenograft model with nanomolar amounts of FOL-CT20p-HBPE-NPs and achieved 100% regression of established tumors as well as prevented tumor growth. These studies indicated that CT20p is a potent and specific anti-cancer agent due to its inhibition of CCT, an essential molecular complex highly expressed in cancer cells, and that the peptide can be efficiently delivered to tumor sites using HBPE-NPs decorated with ligands to receptors, such FR or GRM-1, found on tumor cells. Citation Format: Vishnubhotla P, Khaled AR, Khaled AS, Perez JM, Bassiouni R, Flores O, Nierenberg D. The dynamic duo: A breast cancer-targeting nanoparticle loaded with a cytotoxic peptide as a treatment for metastatic disease. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-02.

Abstract A47: Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer

Abstract The median survival rate for women with metastatic breast cancer is 1-2 years, and the 5-year survival rate is only 15 percent. Early detection of metastatic disease is often ineffective, since once the cancer recurs at a distant site, it may be refractory to conventional treatments. A promising strategy for targeting metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. Immunotherapeutic approaches are effective treatments for many types of cancer, such as melanoma. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. Moreover, immunotherapy can cause adverse side effects such as colitis. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to induce a tumor-specific immune response, which can eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) that in turn activate tumor-specific cytotoxic T cells. While not well-understood, ICD may also sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs, such as anthracyclines, used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of mice with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) which is essential for the folding of actin and tubulin and other critical proteins into their native forms. We found CCT to be overexpressed in breast cancer cells susceptible to the peptide. Inhibition of CCT activity by CT20p, indicated by decreased F-actin, disrupted the cytoskeleton, causing loss of cell motility and adhesion that led to breast cancer cell death. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that can generate the danger signals associate with ICD. To this end, we observed that breast cancer cells treated with CT20p displayed alterations in PERK (protein kinase RNA-like ER kinase), one of the key mediators of the unfolded protein response (UPR). As a result, CT20p-treated breast cancer cells were more readily phagocytosed. This also suggests that peptide treatment could potentially have an impact on NK cell cytotoxicity by inducing the expression of activating ligands on cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p treatment due reduced levels and activity of CCT. These studies indicate that CT20p, by targeting CCT, can be used to induce ICD and stimulate essential innate immune functions like NK cell cytotoxic activity and leukocyte phagocytosis that are necessary for promoting a robust anti-cancer adaptive immune response in order to mitigate the development of lethal metastatic disease. Citation Format: Arati Limaye, Rania Bassiouni, Jeremiah Oyer, Robert W. Igarashi, Orielyz Flores, J. Manuel Perez, Alijca Copik, Annette R. Khaled. Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A47.