N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response.

The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.

m6A Facilitates eIF4F-Independent mRNA Translation.

In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.

Dynamic m(6)A mRNA methylation directs translational control of heat shock response.

The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

O-GlcNAc modification of eIF4GI acts as a translational switch in heat shock response.

Heat shock response (HSR) is an ancient signaling pathway leading to thermoprotection of nearly all living organisms. Emerging evidence suggests that intracellular O-linked ß-N-acetylglucosamine (O-GlcNAc) serves as a molecular 'thermometer' by reporting ambient temperature fluctuations. Whether and how O-GlcNAc modification regulates HSR remains unclear. Here we report that, upon heat shock stress, the key translation initiation factor eIF4GI undergoes dynamic O-GlcNAcylation at the N-terminal region. Without O-GlcNAc modification, the preferential translation of stress mRNAs is impaired. Unexpectedly, stress mRNAs are entrapped within stress granules (SGs) that are no longer dissolved during stress recovery. Mechanistically, we show that stress-induced eIF4GI O-GlcNAcylation repels poly(A)-binding protein 1 and promotes SG disassembly, thereby licensing stress mRNAs for selective translation. Using various eIF4GI mutants created by CRISPR/Cas9, we demonstrate that eIF4GI acts as a translational switch via reversible O-GlcNAcylation. Our study reveals a central mechanism linking heat stress sensing, protein remodeling, SG dynamics and translational reprogramming.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis.

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: -0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

Hsa_circ_0081065 exacerbates IH-induced EndMT via regulating miR-665/HIF-1α signal axis and HIF-1α nuclear translocation.

CircRNAs play an important role in various physiological and pathological biological processes. Despite their widespread involvement, the function of circRNAs in intermittent hypoxia (IH) remain incompletely understood. This study aims to clarify the molecular mechanism of it in IH. Differentially expressed circRNAs were identified by transcriptome sequencing analysis in intermittent hypoxia (IH) model. GO and KEGG enrichment analys were performed on the identified differentially expressed circRNAs. The circular characteristics of hsa_circ_0081065 in human umbilical vein endothelial cells (HUVECs) were detected by RT-qPCR. The sublocalization of hsa_circ_0081065 was examined by FISH. The effect of hsa_circ_0081065 on endothelial to mesenchymal transition (EndMT) was estimated by detecting the expression of EndMT related markers. Various techniques, including RNA-pull down, RIP, EMSA, dual-luciferase reporter assay and immunofluorescence staining were used to investigate the relationship among hsa_circ_0081065, miR-665 and HIF-1α. A total of 13,304 circRNAs were identified in HUVECs treatment with IH, among which 73 were differentially expressed, including 24 upregulated circRNAs and 49 downregulated circRNAs. Notably, hsa_circ_0081065 demonstrated a significantly upregulation. Hsa_circ_0081065 exhibited the circular characteristics of circRNA and was predominantly localized in the cytoplasm. Knockdown of hsa_circ_0081065 inhibited EndMT. Mechanically, we demonstrated that hsa_circ_0081065 acts as a sponge for miR-665 to up-regulate HIF-1α and exacerbate HIF-1α nuclear translocation in HUVECs. We have demonstrated that hsa_circ_0081065 is significantly upregulated in HUVECs treated with IH. Our findings indicate that hsa_circ_0081065 exacerbates IH-induced EndMT through the regulation of the miR-665/HIF-1α signal axis and facilitating HIF-1α nuclear translocation. These results provide a theoretical basis for considering of EndMT as a potential therapeutic target for OSAHS intervention.

Improvement of muscle quality in tilapia (Oreochromis niloticus) with dietary faba bean (Vicia faba L.).

Tilapia (Oreochromis niloticus) is a freshwater fish which is farmed worldwide. Improving the muscle quality of fish has become a major goal while maintaining a sustainable aquaculture system. This research attempts to assess the effect of 0% (FB0), 40%(FB40), 50%(FB50), 60%(FB60) and 70% (FB70) faba bean on the texture parameter, histological analysis, proximate, amino acids, and fatty acids composition in tilapia fed 90 days. The results showed that hardness, chewiness, and shear force of tilapia muscle fed FB60, and FB70 were considerably more in comparison to fish fed FB0 at 90 days (p < 0.05). Tilapia fed faba beans had higher muscle fiber density, wider spaces between muscle fibers and smaller fiber diameter, with the greatest difference in tilapia fed FB60. The total protein content in tilapia fed FB40 was considerably more in comparison to in fish fed FB70 (p < 0.05), where the total protein content in muscle first increased and then reduced with increasing dietary faba bean level. The muscle ∑TAA, ∑EAA, valine, tyrosine, cysteine, aspartic acid, methionine, isoleucine, glutamic acid, leucine, arginine, and serine, contents in tilapia fed FB60 were much more in contrast to in fish fed FB0 (p < 0.05), which initially increased and then reduced with increasing dietary faba bean level. The muscle ∑PUFA content in tilapia fed dietary faba beans was greater compared with fish fed FB0, whereas the ∑SFA contents in tilapia fed FB50 and FB60 were lower in contrast to in fish fed FB0. In summary, dietary faba beans can improve muscle texture, muscle fibers, amino acids content and fatty acids content in tilapia. The research's results make a contribution to the improved knowledge of the association among muscle quality in tilapia and dietary faba beans.

mTORC1 links protein quality and quantity control by sensing chaperone availability.

Balanced protein synthesis and degradation are crucial for proper cellular function. Protein synthesis is tightly coupled to energy status and nutrient levels by the mammalian target of rapamycin complex 1 (mTORC1). Quality of newly synthesized polypeptides is maintained by the molecular chaperone and ubiquitin-proteasome systems. Little is known about how cells integrate information about the quantity and quality of translational products simultaneously. We demonstrate that cells distinguish moderate reductions in protein quality from severe protein misfolding using molecular chaperones to differentially regulate mTORC1 signaling. Moderate reduction of chaperone availability enhances mTORC1 signaling, whereas stress-induced complete depletion of chaperoning capacity suppresses mTORC1 signaling. Molecular chaperones regulate mTORC1 assembly in coordination with nutrient availability. This mechanism enables mTORC1 to rapidly detect and respond to environmental cues while also sensing intracellular protein misfolding. The tight linkage between protein quality and quantity control provides a plausible mechanism coupling protein misfolding with metabolic dyshomeostasis.

Baseline characteristics and changes of biomarkers in disease course predict prognosis of patients with COVID-19.

The outbreak of coronavirus disease (COVID-19) has brought great challenges to the world. The objectives of this study were to describe the baseline characteristics and changes of biomarkers of these COVID-19 patients and identify predictive value of the above markers for patient death. Using patient death as the observational endpoints, clinical data of inpatients in a special ward for COVID-19 in Wuhan, China were retrospectively collected. Univariate and multivariate Cox regression analyses were used to evaluate prognostic value of baseline characteristics and laboratory data changes. This study included clinical data of 75 patients. Age, c-reactive protein (CRP) and interleukin-6 levels were independent predictors of patient death. Survivors were characterized as having declining neutrophil counts, D-dimer, N-terminal pronatriuretic peptide, troponin I (TnI) and c-reactive protein levels, while counts of lymphocyte gradually came back. Non-survivors were characterized with increasing white blood cell counts (WBC) and neutrophil counts. Changes of WBC, TnI and interleukin-6 were also independently associated with patient death. Older age, baseline CRP and IL-6 levels may be used as meaningful predictors to identify patients with poor prognosis. Changes of biomarkers should be closely monitored in the management of patients with COVID-19, while constantly increasing levels of WBC, TnI and interleukin-6 in the disease course also predict patient death.

Abnormal liver-related biomarkers in COVID-19 patients and the role of prealbumin.

Background/Aims: We aimed to evaluate the distribution of abnormal liver-related biomarkers in patients with coronavirus disease (COVID-19) and explore the prognostic value of elevated liver enzymes and abnormal liver synthetic capacity with regards to patient mortality. Patients and Methods: This retrospective observational study included 80 laboratory-confirmed COVID-19 cases. Data were collected from the electronic medical record system by a trained team of physicians. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), albumin, and prealbumin levels at admission and on day 7 after admission were collected. The primary outcome of the current study was patient mortality. Results: Abnormal ALT, AST, TB, albumin, and prealbumin levels were observed in 11 (13.8%), 15 (18.8%), 5 (6.3%), 22 (27.5%), and 31 (38.8%) patients, respectively. Male gender correlated with elevated ALT and AST levels (p = 0.027 and 0.036, respectively). Higher levels of AST and lower levels of albumin and prealbumin were associated with patient mortality (p = 0.009, 0.002, and 0.003, respectively). Multivariate Cox regression analysis identified patient age (p = 0.013, HR 1.108) and prealbumin levels (p = 0.015, HR 0.986) as independent predictors for patient mortality. However, changes in liver-related biomarkers were not associated with poor outcome in multivariate analysis (p > 0.05). Conclusions: Abnormalities in albumin and prealbumin levels are common among COVID-19 patients and hypoprealbuminemia independently predicts adverse outcome and should be carefully considered in clinical practice. Moreover, changes in liver-related biomarkers is not a salient feature of COVID-19.

Co-expression of P1A35-43/beta2m fusion protein and co-stimulatory molecule CD80 elicits effective anti-tumor immunity in the P815 mouse mastocytoma tumor model.

A strong CTL response is dependent upon a high level of expression of specific class I major histocompatibility complex (MHC)/peptide complexes at the cell surface. An epitope-linked beta2-microglobulin (beta2m) molecule could provide a simple and more efficient means to enhance the formation of defined MHC/peptide complexes. However, the ability of an epitope-linked beta2m molecule to elicit primary CTL responses in vivo is still unknown. In this study, we modified the P1A tumor cell vaccine by addition of the tumor-associated epitope (TAE)-linked beta2m molecule and co-stimulatory molecule CD80 to improve the efficiency in the application of the vaccine. A eukaryotic co-expression vector consisting of the P1A35-43-linked beta2m molecule and the murine CD80 gene was constructed. P815 cell lines stably expressing P1A35-43-linked beta2m molecule and/or CD80 were established after transfection, by selection under G418. Administration of these inactivated tumor cell vaccines allowed the TAE-specific CD8+ T cell responses to be examined in vivo. Our results indicate that immunization with P815 cells expressing both the P1A35-43-linked beta2m molecule and the murine CD80 gene elicited a significantly stronger antitumor immune response than the single-modified tumor cell vaccines (expressing either P1A35-43-linked beta2m or CD80 alone). These findings support the feasibility and effectiveness of developing a dual-modified tumor cell vaccine consisting of the epitope-linked beta2m molecule and a co-stimulatory molecule.

A study on anti-tumor immunity induced by gene-modified melanoma B16 cells.

T cell-mediated cell immunity is the main anti-tumor immunity in which the effector T cells need specific antigen and costimulatory signals. One of the vaccines applied in tumor immunotherapy is the gene-modified tumor cell vaccine. One potential method to increase cell epitope density is to link the antigen with the major histcompatibility complex subunit beta2m. Our previous research indicated that the strategy of epitope fusion gene OVA-linker-beta2m can promote the formation of specific compounds on the tumor cell surface in vitro. In this study, we constructed two coexpression vectors pGL3-CD80-OVA-linker-beta2m and pGL3-IL21-OVA-linker-beta2m, in order to explore the cooperative action of CD80 or interleukin-21 (IL21) with the epitope fusion gene in anti-tumor immunity. Results showed that gene-modified B16 cells (B16/OVA, B16/CD80-OVA and B16/IL21-OVA) grew slower than B16 cells in vitro and in vivo, especially the B16/IL21-OVA subline, which illustrated that such gene modification decreased oncogenicity of malignant tumor cells. On the other hand, gene-modified tumor cell subline immunization can induce effective long-term anti-tumor immunity defending tumor cell attacks. IL21 played a more cooperative role with the OVA-linker-beta2m than CD80 in this study. This strategy might lay foundations for the research of a new type of tumor vaccine.

A Coding Sequence-Embedded Principle Governs Translational Reading Frame Fidelity.

Upon initiation at a start codon, the ribosome must maintain the correct reading frame for hundreds of codons in order to produce functional proteins. While some sequence elements are able to trigger programmed ribosomal frameshifting (PRF), very little is known about how the ribosome normally prevents spontaneous frameshift errors that can have dire consequences if uncorrected. Using high resolution ribosome profiling data sets, we discovered that the translating ribosome uses the 3' end of 18S rRNA to scan the AUG-like codons after the decoding process. The postdecoding mRNA:rRNA interaction not only contributes to predominant translational pausing, but also provides a retrospective mechanism to safeguard the ribosome in the correct reading frame. Partially eliminating the AUG-like "sticky" codons in the reporter message leads to increased +1 frameshift errors. Remarkably, mutating the highly conserved CAU triplet of 18S rRNA globally changes the codon "stickiness". Further supporting the role of "sticky" sequences in reading frame maintenance, the codon composition of open reading frames is highly optimized across eukaryotic genomes. These results suggest an important layer of information embedded within the protein-coding sequences that instructs the ribosome to ensure reading frame fidelity during translation.

The effect of co-expression costimulatory molecule CD80 on uptake of antigen peptide-MHC class I-GFP complex by specific T cells.

CD80, a costimulatory molecule, plays an important role in eliciting antitumor immunity. Without costimulation, recognition of antigens by T cells may not cause a response, even if tumor cells express MHC class I molecules and specific antigens. On the basis of the recombinant GFP-tagged Kb molecule, we constructed a co-expression vector of CD80 and GFP-tagged Kb molecules. The recombinant fusion was transfected into mouse melanoma B16 cells by electroporation; positive cells were obtained by G418 screening. Highly expressing monoclonal cells, irradiated by 137Cs, were used to immunize mice to obtain specific T cells, which were then cultivated with tumor cells in vitro and examined with a laser confocal microscope. The evident and intense uptake of the antigen peptide-MHC class I-GFP complex by specific T cells was visualized from the culture of B16/CD80-Kb-GFP and T cells. However, little uptake was observed from the culture of B16/Kb-GFP and T cells. These results show that co-expression of CD80 molecules with Kb, an MHC class I molecule, on the surface of B16 tumor cells can enhance the response of specific T cells and thus increase the uptake of the antigen peptide-MHC class I-GFP complex. The absorbed green fluorescence was concentrated mainly on the T cell surface, and this result might pave the way to eluting specific antigen peptides directly from T cells to find and isolate novel tumor-specific antigen peptides.

[Inhibition of VEGF expression by plasmid-based RNA interference in the retinoblastoma cells].

OBJECTIVE: To explore the inhibition effect of small hairpin RNA (shRNA) expression plasmid targeting vascular endothelial growth factor (VEGF) on VEGF expression in cultured retinoblastoma (RB) cells. METHODS: VEGF shRNA plasmid p4.1CMV-VEGF was constructed and transfected into retinoblastoma cell lines SO-RB50 and HXO-RB44. Using neomycin G418 in conjunction with gradient dilution, p4.1CMV-VEGF shRNA positive single clone of RB cells was selected and subsequently enriched. Real-time polymerase chain reaction (PCR) was applied to detect VEGF mRNA levels of RB cells. VEGF protein concentration in culture supernatants of RB cells were determined by enzyme-linked immunosorbent assay (ELISA). Plasmid p4.1CMV-Neg shRNA, which expressed shRNA lacking significant sequence identity to human and mouse genome databases, was transfected into RB cells as negative control. Cells without any treatment were used as blank controls. RESULTS: p4.1CMV-VEGF shRNA was constructed successfully and VEGF shRNA construct positive clone of RB cells was developed. VEGF mRNA level of SO-RB50 (HXO-RB44) cells in negative control and blank control was 5.02 (5.70) folds and 6.32 (4.86) folds greater than that in p4.1CMV-VEGF shRNA treated SO-RB50 (HXO-RB44) cells. VEGF protein concentration in culture supernatants of p4.1CMV-VEGF shRNA treated SO-RB50 cells (187.69 +/- 83.89) microg/L was significantly lower than that of negative control (822.98 +/- 187.98) microg/L and blank control (865.76 +/- 170.33) microg/L (P < 0.01). There was also significant difference of VEGF protein concentration between p4.1CMV-VEGF shRNA treated HXO-RB44 cells (162.20 +/- 66.33) microg/L and controls (764.33 +/- 164.79) microg/L in negative control and (828.22 +/- 145.94) microg/L in blank control (P < 0.01). CONCLUSIONS: Stable transfection of VEGF shRNA expression plasmid can potently suppress VEGF expression in RB cells. RNA interference (RNAi) targeting VEGF promises to be a substantial tool for the study of the treatment of RB.

Translational control of the cytosolic stress response by mitochondrial ribosomal protein L18.

In response to stress, cells attenuate global protein synthesis but permit efficient translation of mRNAs encoding heat-shock proteins (HSPs). Although decades have passed since the first description of the heat-shock response, how cells achieve translational control of HSP synthesis remains enigmatic. Here we report an unexpected role for mitochondrial ribosomal protein L18 (MRPL18) in the mammalian cytosolic stress response. MRPL18 bears a downstream CUG start codon and generates a cytosolic isoform in a stress-dependent manner. Cytosolic MRPL18 incorporates into the 80S ribosome and facilitates ribosome engagement on mRNAs selected for translation during stress. MRPL18 knockdown has minimal effects on mitochondrial function but substantially dampens cytosolic HSP expression at the level of translation. Our results uncover a hitherto-uncharacterized stress-adaptation mechanism in mammalian cells, which involves formation of a 'hybrid' ribosome responsible for translational regulation during the cytosolic stress response.

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation.

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes.

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.