Unzipping the Secrets of Amyloid Disassembly by the Human Disaggregase

Neurodegenerative diseases (NDs) are increasingly positioned as leading causes of global deaths. The accelerated aging of the population and its strong relationship with neurodegeneration forecast these pathologies as a huge global health problem in the upcoming years. In this scenario, there is an urgent need for understanding the basic molecular mechanisms associated with such diseases. A major molecular hallmark of most NDs is the accumulation of insoluble and toxic protein aggregates, known as amyloids, in extracellular or intracellular deposits. Here, we review the current knowledge on how molecular chaperones, and more specifically a ternary protein complex referred to as the human disaggregase, deals with amyloids. This machinery, composed of the constitutive Hsp70 (Hsc70), the class B J-protein DnaJB1 and the nucleotide exchange factor Apg2 (Hsp110), disassembles amyloids of α-synuclein implicated in Parkinson’s disease as well as of other disease-associated proteins such as tau and huntingtin. We highlight recent studies that have led to the dissection of the mechanism used by this chaperone system to perform its disaggregase activity. We also discuss whether this chaperone-mediated disassembly mechanism could be used to solubilize other amyloidogenic substrates. Finally, we evaluate the implications of the chaperone system in amyloid clearance and associated toxicity, which could be critical for the development of new therapies.

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+ ] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+ ] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for propagation of what otherwise would be lethal [PSI+] prions.

Dual topology of co-chaperones at the membrane of the endoplasmic reticulum

Dual topologies of proteins at the ER membrane are known for a variety of proteins allowing the same protein to exert different functions according to the topology adopted. A dual topology of the co-chaperone ERdj4, which resides in the endoplasmic reticulum (ER), was proposed recently, a thesis that we found to align all published data and existing controversies into one whole picture. The aim of this review is to reassess all primary data available in the literature on ER-resident Hsp40 co-chaperones with respect to their topology. After careful and critical analyses of all experimental data published so far, we identified, next to ERdj4, two other co-chaperones, ERdj3 and ERdj6, that also display features of a dual topology at the ER membrane. We assume that during cellular stress subpools of some ER-resident J protein can alter their topology so that these proteins can exert different functions in order to adapt to cellular stress.

Deciphering Network Crosstalk: The Current Status and Potential of miRNA Regulatory Networks on the HSP40 Molecular Chaperone Network

Molecular chaperone networks fulfill complex roles in protein homeostasis and are essential for maintaining cell health. Hsp40s (commonly referred to as J-proteins) have critical roles in development and are associated with a variety of human diseases, yet little is known regarding the J-proteins with respect to the post-transcriptional mechanisms that regulate their expression. With relatively small alterations in their abundance and stoichiometry altering their activity, post-transcriptional regulation potentially has significant impact on the functions of J-proteins. MicroRNAs (miRNAs) are a large group of non-coding RNAs that form a complex regulatory network impacting gene expression. Here we review and investigate the current knowledge and potential intersection of miRNA regulatory networks with the J-Protein chaperone network. Analysis of datasets from the current version of TargetScan revealed a great number of predicted microRNAs targeting J-proteins compared to the limited reports of interactions to date. There are likely unstudied regulatory interactions that influence chaperone biology contained within our analysis. We go on to present some criteria for prioritizing candidate interactions including potential cooperative targeting of J-Proteins by multiple miRNAs. In summary, we offer a view on the scope of regulation of J-Proteins through miRNAs with the aim of guiding future investigations by identifying key regulatory nodes within these two complex cellular networks.

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

Differential roles for DNAJ isoforms in HTT-polyQ and mutant FUS aggregation modulation revealed by chaperone network screens

Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntington’s disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completely different than those of HTT-polyQ. Interestingly, different naturally-occurring isoforms of DNAJ chaperones had opposing effects on HTT-polyQ vs. mutFUS aggregation. We identified a complex of the full length (FL) DNAJB14 and DNAJB12 isoforms which substantially alleviated mutFUS aggregation, in an HSP70-dependent manner. Their naturally occurring short isoforms were unable to form the complex, nor to interact with HSP70, and lost their ability to reduce mutFUS aggregation. In contrast, the short isoform of DNAJB12 significantly alleviated HTT-polyQ aggregation, while DNAJB12-FL aggravated HTT-polyQ aggregation. Finally, we demonstrated that full-length DNAJB14 ameliorated mutFUS aggregation compared to DNAJB14-short in primary neurons. Together, our data unraveled distinct molecular properties required for aggregation protection in different neurodegenerative diseases, and revealed a new layer of complexity of the chaperone network elicited by naturally occurring J-protein isoforms, highlighting functional diversity among the DNAJ family.

Subcellular localization of the J-protein Sis1 regulates the heat shock response

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Subcellular localization of the J-protein Sis1 regulates the heat shock response

ABSTRACTCells exposed to heat shock induce a conserved gene expression program – the heat shock response (HSR) – encoding chaperones like Hsp70 and other protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1 – a co-chaperone for Hsp70 – controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the cortical ER. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.One sentence summaryLocalization of the J-protein Sis1 to a subcellular network of proteostasis factors activates the heat shock response.

Arabidopsis chloroplast J protein DJC75/CRRJ mediates nitrate-promoted seed germination in the dark

Background and Aims Nitrate can stimulate seed germination of many plant species in the absence of light; however, the molecular mechanism of nitrate-promoted seed germination in the dark remains largely unclear and no component of this pathway has been identified yet. Here, we show that a plastid J-domain protein, DJC75/CRRJ, in arabidopsis (Arabidopsis thaliana) is important for nitrate-promoted seed germination in the dark. Methods The expression of DJC75 during imbibition in the dark was investigated. The seed germination rate of mutants defective in DJC75 was determined in the presence of nitrate when light cues for seed germination were eliminated by the treatment of imbibed seeds with a pulse of far-red light to inactivate phytochrome B (phyB), or by assaying germination in the dark with seeds harbouring the phyB mutation. The germination rates of mutants defective in CRRL, a J-like protein related to DJC75, and in two chloroplast Hsp70s were also measured in the presence of nitrate in darkness. Key Results DJC75 was expressed during seed imbibition in the absence of light. Mutants defective in DJC75 showed seed germination defects in the presence of nitrate when light cues for seed germination were eliminated. Mutants defective in CRRL and in two chloroplast Hsp70s also exhibited similar seed germination defects. Upregulation of gibberellin biosynthetic gene GA3ox1 expression by nitrate in imbibed phyB mutant seeds was diminished when DJC75 was knocked out. Conclusions Our data suggest that plastid J-domain protein DJC75 regulates nitrate-promoted seed germination in the dark by upregulation of expression of the gibberellin biosynthetic gene GA3ox1 through an unknown mechanism and that DJC75 may work in concert with chloroplast Hsp70s to regulate nitrate-promoted seed germination. DJC75 is the first pathway component identified for nitrate-promoted seed germination in the dark.

Homologue replacement in the import motor of the mitochondrial inner membrane of trypanosomes

Many mitochondrial proteins contain N-terminal presequences that direct them to the organelle. The main driving force for their translocation across the inner membrane is provided by the presequence translocase-associated motor (PAM) which contains the J-protein Pam18. Here, we show that in the PAM of Trypanosoma brucei the function of Pam18 has been replaced by the non-orthologous euglenozoan-specific J-protein TbPam27. TbPam27 is specifically required for the import of mitochondrial presequence-containing but not for carrier proteins. Similar to yeast Pam18, TbPam27 requires an intact J-domain to function. Surprisingly, T. brucei still contains a bona fide Pam18 orthologue that, while essential for normal growth, is not involved in protein import. Thus, during evolution of kinetoplastids, Pam18 has been replaced by TbPam27. We propose that this replacement is linked to the transition from two ancestral and functionally distinct TIM complexes, found in most eukaryotes, to the single bifunctional TIM complex present in trypanosomes.