Maintain Protein Homeostasis Research Articles

Endoplasmic reticulum stress in acute lung injury and pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive and irreversible lung disease that leads to diminished lung function, respiratory failure, and ultimately death and typically has a poor prognosis, with an average survival time of 2 to 5 years. Related articles suggested that endoplasmic reticulum (ER) stress played a critical role in the occurrence and progression of PF. The ER is responsible for maintaining protein homeostasis. However, factors such as aging, hypoxia, oxidative stress, or inflammation can disrupt this balance, promoting the accumulation of misfolded proteins in the ER and triggering ER stress. To cope with this situation, cells activate the unfolded protein response (UPR). Since acute lung injury (ALI) is one of the key onset events of PF, in this review, we will discuss the role of ER stress in ALI and PF by activating multiple signaling pathways and molecular mechanisms that affect the function and behavior of different cell types, with a focus on epithelial cells, fibroblasts, and macrophages. Linking ER stress to these cell types may broaden our understanding of the mechanisms underlying lung fibrosis and help us target these cells through these mechanisms. The relationship between ER stress and PF is still evolving, and future research will explore new strategies to regulate UPR pathways, providing novel therapeutic targets.

Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.

The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.

SUMO-targeted Ubiquitin Ligases as crucial mediators of protein homeostasis in Candida glabrata.

Candida glabrata is an opportunistic human pathogen, capable of causing severe systemic infections that are often resistant to standard antifungal treatments. To understand the importance of protein SUMOylation in the physiology and pathogenesis of C. glabrata, we earlier identified the components of SUMOylation pathway and demonstrated that the deSUMOylase CgUlp2 is essential for pathogenesis. In this work we show that the CgUlp2 is essential to maintain protein homeostasis via the SUMO-targeted ubiquitin ligase pathway. The dual loss of deSUMOylase and specific ubiquitin ligase, CgSlx8, results in heightened protein degradation, rendering the cells vulnerable to various stressors. This degradation affects crucial processes such as purine biosynthesis and compromises mitochondrial function in the mutants. Importantly, the absence of these ubiquitin ligases impedes the proliferation of C. glabrata in macrophages. These findings underscore the significance of SUMOylation and SUMO-mediated protein homeostasis as pivotal regulators of C. glabrata physiology and capacity to survive in host cells. Understanding these mechanisms could pave the way for the development of effective antifungal treatments.

The Greatwall-Endosulfine-PP2A/B55 pathway regulates entry into quiescence by enhancing translation of Elongator-tunable transcripts

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. In this study, we demonstrate that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications crucial for sustaining the translation programme during entry into quiescence. This mechanism promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, several of these mRNAs encode TORC1 inhibitors, TORC2 activators, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between tRNA epitranscriptome and biased codon usage.

Identification of a factor that accelerates substrate release from the signal recognition particle.

The eukaryotic signal recognition particle (SRP) cotranslationally recognizes the first hydrophobic segment of nascent secretory and membrane proteins and delivers them to a receptor at the endoplasmic reticulum (ER). How substrates are released from SRP at the ER to subsequently access translocation factors is not well understood. We found that TMEM208 can engage the substrate binding domain of SRP to accelerate release of its bound cargo. Without TMEM208, slow cargo release resulted in excessive synthesis of downstream polypeptide before engaging translocation factors. Delayed access to translocation machinery caused progressive loss of insertion competence, particularly for multipass membrane proteins, resulting in their impaired biogenesis. Thus, TMEM208 facilitates prompt cargo handover from the targeting to translocation machinery to minimize biogenesis errors and maintain protein homeostasis.

Neuronal fatty acid-binding protein enhances autophagy and suppresses amyloid-β pathology in a Drosophila model of Alzheimer's disease.

Fatty acid-binding proteins (FABPs) are small cytoplasmic proteins involved in intracellular lipid transport and bind free fatty acids, cholesterol, and retinoids. FABP3, the major neuronal FABP in the adult brain, is upregulated in the CSF of patients with Alzheimer's disease (AD). However, the precise role of neuronal FABPs in AD pathogenesis remains unclear. This study investigates the contribution of fabp, the Drosophila homolog of FABP3 and FABP7, to amyloid β (Aβ) pathology using a Drosophila model. Neuronal knockdown of fabp shortened the lifespan of flies and increased age-related protein aggregates in the brain. In an AD model, fabp knockdown in neurons increased Aβ accumulation and Aβ-induced neurodegeneration, whereas fabp overexpression ameliorated Aβ pathology. Notably, fabp overexpression stimulated autophagy, which was inhibited by the knockdown of Eip75B, the Drosophila homolog of the peroxisome proliferator-activated receptor (PPAR). The PPAR activator rosiglitazone restored autophagy impaired by fabp knockdown and reduced fabp knockdown-induced increased Aβ aggregation and cell death. Furthermore, knockdown of either fabp or Eip75B in the wing imaginal disc or adult fly brain reduced the expression of Atg6 and Atg8a. Additionally, treatment of the fabp knockdown AD model flies with polyunsaturated fatty acids, such as docosahexaenoic acid or linoleic acid, partially alleviated cell death in the developing eye, restored impaired autophagy flux, reduced Aβ aggregation, and attenuated Aβ-induced cell death. Our results suggest that Drosophila fabp plays an important role in maintaining protein homeostasis during aging and protects neurons from Aβ-induced cell death by enhancing autophagy through the PPAR pathway. These findings highlight the potential importance of neuronal FABP function in AD pathogenesis.

Selective protein degradation through chaperone‑mediated autophagy: Implications for cellular homeostasis and disease (Review).

Cells rely on autophagy for the degradation and recycling of damaged proteins and organelles. Chaperone-mediated autophagy (CMA) is a selective process targeting proteins for degradation through the coordinated function of molecular chaperones and the lysosome‑associated membrane protein‑2A receptor (LAMP2A), pivotal in various cellular processes from signal transduction to the modulation of cellular responses under stress. In the present review, the intricate regulatory mechanisms of CMA were elucidated through multiple signaling pathways such as retinoic acid receptor (RAR)α, AMP‑activated protein kinase (AMPK), p38‑TEEB‑NLRP3, calcium signaling‑NFAT and PI3K/AKT, thereby expanding the current understanding of CMA regulation. A comprehensive exploration of CMA's versatile roles in cellular physiology were further provided, including its involvement in maintaining protein homeostasis, regulating ferroptosis, modulating metabolic diversity and influencing cell cycle and proliferation. Additionally, the impact of CMA on disease progression and therapeutic outcomes were highlighted, encompassing neurodegenerative disorders, cancer and various organ‑specific diseases. Therapeutic strategies targeting CMA, such as drug development and gene therapy were also proposed, providing valuable directions for future clinical research. By integrating recent research findings, the present review aimed to enhance the current understanding of cellular homeostasis processes and emphasize the potential of targeting CMA in therapeutic strategies for diseases marked by CMA dysfunction.

HSF1/HSP25 system protects mitochondria function from heat stress and assists steroidogenesis in MA-10 Leydig cells

Heat shock response is characterized by the induction of heat shock proteins (HSPs) or molecular chaperones that maintain protein homeostasis. Heat shock transcription factor 1 (HSF1) plays a central role in heat shock response in mammalian cells. To investigate the impact of the heat shock response mechanism on steroidogenesis, we generated MA-10 mouse Leydig tumor cells deficient in HSF1 using CRISPR-Cas9 genome editing. Under heat stress conditions, the levels of StAR protein, but not its mRNA, decreased more in HSF1-knockout cells than in wild-type cells, confirming that HSF1 stabilizes StAR protein. Simultaneously, HSP110, HSP70, and HSP25 were markedly upregulated in a manner dependent on HSF1. Mitochondrial membrane potential (MMP) and ATP synthesis were decreased in HSF1-knockout cells under heat stress conditions, and mitochondrial fragmentation was enhanced. Furthermore, treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a disruptor of MMP, reduced the levels of StAR protein to a greater extent in HSF1-knockout cells than in wild-type cells, which was associated with decreased MMP and ATP synthesis. Unexpectedly, HSP25 expression was markedly increased in wild-type cells following CCCP treatment. HSP25 knockdown reduces MMP under heat stress conditions and decreases StAR protein levels and progesterone synthesis. HSP25 overexpression in HSF1KO cells restored StAR protein levels. These results show that the HSF1/HSP25 pathway protects mitochondrial function and maintains StAR synthesis.

Targeting Protein Aggregation in ALS.

Proteinopathies involve the abnormal accumulation of specific proteins. Maintaining the balance of the proteome is a finely regulated process managed by a complex network of cellular machinery responsible for protein synthesis, folding, and degradation. However, stress and ageing can disrupt this balance, leading to widespread protein aggregation. Currently, several therapies targeting protein aggregation are in clinical trials for ALS. These approaches mainly focus on two strategies: addressing proteins that are prone to aggregation due to mutations and targeting the cellular mechanisms that maintain protein homeostasis to prevent aggregation. This review will cover these emerging drugs. Advances in ALS research not only offer hope for better outcomes for ALS patients but also provide valuable insights and methodologies that can benefit the broader field of neurodegenerative disease drug discovery.

Adenosine Triphosphate Inhibits Cold-Responsive Aggregation.

Adenosine triphosphate (ATP), ubiquitous in all living organisms, is conventionally recognized as a fundamental energy currency essential for a myriad of cellular processes. While its traditional role in energy metabolism requires only micromolar concentrations, the cellular content of ATP has been found to be significantly higher at the millimolar level. Recent studies have attempted to correlate this higher concentration of ATP with its nonenergetic role in maintaining protein homeostasis, leaving the investigation of ATP's nontrivial activities in biology an open question. Here, by coupling computer simulations and experiments, we uncover new insights into ATP's role as a cryoprotectant against cold-salt stress, highlighting the necessity for higher cellular ATP concentrations. We present direct evidence at charged silica interfaces, demonstrating ATP's ability to restore native intersurface interactions disrupted by combined cold-salt stress, thereby inhibiting cold-responsive aggregation in high-salt conditions. ATP desorbs salt cations from negatively charged surfaces through predominant interactions between ATP and the salt cations. Although the mode of ATP's action remains unchanged with temperature, the extent of interaction scales with temperature, requiring less ATP activity at lower temperatures, justifying the reason for reduction in cellular ATP content due to the cold effect, reported in previous experimental studies. The trend observed in inorganic nanostructures is recurrent and robustly transferable to charged protein interfaces. A thorough comparison of ATP's cryoprotective activity with traditionally known biological cryoprotectants (glycine and betaine) reveals ATP's greater efficiency. In retrospect, our findings highlight ATP's additional biological role in cryopreservation, expanding its potential biomedical applications by offering effective protection of cells from cryoinjuries and avoiding the significant challenges associated with the toxicity of organic cryoprotectants.

Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis

Ubiquitination is crucial for maintaining protein homeostasis and plays a vital role in diverse biological processes. Ubiquitinome profiling and quantification are of great scientific significance. Artificial ubiquitin-binding domains (UBDs) have been widely employed to capture ubiquitinated proteins. The success of this enrichment relies on recognizing native spatial structures of ubiquitin and ubiquitin chains by UBDs under native conditions. However, the use of native lysis conditions presents significant challenges, including insufficient protein extraction, heightened activity of deubiquitinating enzymes and proteasomes in removing the ubiquitin signal, and purification of a substantial number of contaminant proteins, all of which undermine the robustness and reproducibility of ubiquitinomics. In this study, we introduced a novel approach that combines denatured-refolded ubiquitinated sample preparation (DRUSP) with a tandem hybrid UBD for ubiquitinomic analysis. The samples were effectively extracted using strongly denatured buffers and subsequently refolded using filters. DRUSP yielded a significantly stronger ubiquitin signal, nearly three times greater than that of the Control method. Then, eight types of ubiquitin chains were quickly and accurately restored; therefore, they were recognized and enriched by tandem hybrid UBD with high efficiency and no biases. Compared with the Control method, DRUSP showed extremely high efficiency in enriching ubiquitinated proteins, improving overall ubiquitin signal enrichment by approximately 10-fold. Moreover, when combined with ubiquitin chain-specific UBDs, DRUSP had also been proven to be a versatile approach. This new method significantly enhanced the stability and reproducibility of ubiquitinomics research. Finally, DRUSP was successfully applied to deep ubiquitinome profiling of early mouse liver fibrosis with increased accuracy, revealing novel insights for liver fibrosis research.

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this "cataract-reversing" phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress-related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

Unveiling the Intercompartmental Signaling Axis: Mitochondrial to ER Stress Response (MERSR) and its Impact on Proteostasis.

Maintaining protein homeostasis is essential for cellular health. Our previous research uncovered a cross-compartmental Mitochondrial to Cytosolic Stress Response, activated by the perturbation of mitochondrial proteostasis, which ultimately results in the improvement of proteostasis in the cytosol. Here, we found that this signaling axis also influences the unfolded protein response of the endoplasmic reticulum (UPR ER ), suggesting the presence of a Mitochondria to ER Stress Response (MERSR). During MERSR, the IRE1 branch of UPR ER is inhibited, introducing a previously unknown regulatory component of MCSR. Moreover, proteostasis is enhanced through the upregulation of the PERK-eIF2α signaling pathway, increasing phosphorylation of eIF2α and improving the ER's ability to handle proteostasis. MERSR activation in both polyglutamine and amyloid-beta peptide-expressing C. elegans disease models also led to improvement in both aggregate burden and overall disease outcome. These findings shed light on the coordination between the mitochondria and the ER in maintaining cellular proteostasis and provide further evidence for the importance of intercompartmental signaling.

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of ‘cell cycle genes’, which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Pharmacologic activation of activating transcription factor 6 contributes to neuronal survival after spinal cord injury in mice.

The impact of primary and secondary injuries of spinal cord injury (SCI) results in the demise of numerous neurons, and there is still no efficacious pharmacological intervention for it. Recently, studies have shown that endoplasmic reticulum stress (ERS) plays a pivotal role in recovery of neurological function after spinal cord injury. As a process to cope with intracellular accumulation of misfolded and unfolded proteins which triggers ERS, the unfolded protein response (UPR) plays an important role in maintaining protein homeostasis. And, a recently disclosed small molecule AA147, which selectively activates activating transcription factor 6 (ATF6), has shown promising pharmacological effects in several disease models. Thus, it seems feasible to protect the neurons after spinal cord injury by modulating UPR. In this study, primary neurons were isolated from E17-19 C57BL/6J mouse embryos and we observed that AA147 effectively promoted the survival of neurons and alleviated neuronal apoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R) invitro. This was evident through a decrease in the proportion of PI-positive and TUNEL-positive cells, an increase in BCL-2 expression, and a decrease in the expression of BAX and C-caspase3. In in-vivo experiments, these findings were corroborated by TUNEL staining and immunohistochemistry. It was also found that AA147 enhanced three arms of the unfolded protein response with reduced CHOP expression. Besides, AA147 mitigated the accumulation of ROS in neurons probably by upregulating catalase expression. Furthermore, spinal cord injury models of C57BL/6J mice were established and behavioral experiments revealed that AA147 facilitated the recovery of motor function following SCI. Thus, pharmacologic activation of ATF6 represents a promise therapeutic approach to ameliorate the prognosis of SCI.

The Role of Protein Quantity Control in Polyglutamine Spinocerebellar Ataxias.

Polyglutamine spinocerebellar ataxias (polyQ SCAs) represent the most prevalent subtype of SCAs. The primary pathogenic mechanism is believed to be the gain-of-function neurotoxicity of polyQ proteins. Strategies such as enhancing the degradation or inhibiting the accumulation of these mutant proteins are pivotal for reducing their toxicity and slowing disease progression. The protein quality control (PQC) system, comprising primarily molecular chaperones and the ubiquitin‒proteasome system (UPS), is essential for maintaining protein homeostasis by regulating protein folding, trafficking, and degradation. Notably, polyQ proteins can disrupt the PQC system by sequestering its critical components and impairing its proteasomal functions. Therefore, restoring the PQC system through genetic or pharmacological interventions could potentially offer beneficial effects and alleviate the symptoms of the disease. Here, we will provide a review on the distribution, expression, and genetic or pharmacological intervention of protein quality control system in cellular or animal models of PolyQ SCAs.

Actin-bundling protein fimbrin serves as a new auxin biosynthesis orchestrator in Arabidopsis root tips.

Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well-established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC-MS/MS-based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton-26S proteasome-YUC8-auxin' axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.

Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system.

The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.