DNAJC15 Human

What is DNAJC15 and what are its key molecular characteristics?

DNAJC15 (DnaJ homolog subfamily C member 15) is a mitochondrial protein of approximately 16 kDa that functions as a negative regulator of the mitochondrial respiratory chain. It prevents mitochondrial hyperpolarization and restricts ATP generation . DNAJC15 is also known by alternative names including MCJ, DNAJD1, GIG22, and HSD18 . Structurally, it contains a transmembrane domain anchoring it to the inner mitochondrial membrane, with its characteristic J-domain facing the matrix where it stimulates the ATPase activity of HSPA9 . Unlike its paralog DNAJC19, DNAJC15 possesses an extended amino-terminal region exposed to the intermembrane space that can be cleaved by OMA1 protease .

What is the primary role of DNAJC15 in mitochondrial function?

DNAJC15 serves dual roles in mitochondrial biology. First, it acts as an import component of the TIM23 translocase complex, facilitating the import of nuclear-encoded proteins into the mitochondrial matrix and inner membrane . Second, DNAJC15 regulates mitochondrial respiration by functioning as a negative regulator of the respiratory chain under normal conditions . These roles position DNAJC15 at the intersection of mitochondrial protein import, quality control, and bioenergetics, making it a critical coordinator of mitochondrial function.

What are validated methods for detecting and quantifying DNAJC15?

For protein detection, Western blotting using mouse polyclonal antibodies (e.g., ab167199) at 1 μg/mL concentration is effective, with an expected band size of 16 kDa . When performing Western blots, include positive controls (such as DNAJC15-transfected 293T cells) and negative controls (non-transfected cells) . For mRNA analysis, quantitative RT-PCR or Northern blotting can be employed. In mice, DNAJC15 mRNA is highly abundant in heart tissue, which can serve as a positive control for expression studies .

What strategies are effective for manipulating DNAJC15 expression in experimental systems?

For loss-of-function studies, both transient siRNA-mediated knockdown and stable CRISPR-Cas9 genome editing have been successfully employed to generate DNAJC15-depleted cells . When designing these experiments, consider the functional redundancy between DNAJC15 and DNAJC19, as simultaneous depletion of both proteins has more profound effects on cell proliferation than single knockdowns . For gain-of-function approaches, transfection with expression vectors containing full-length DNAJC15 cDNA or specific variants (DNAJC15D2 or S-DNAJC15) has been effective in rescue experiments . These genetic manipulation approaches allow for detailed analysis of DNAJC15's functions in various cellular processes.

How can the DNAJC15 interactome be comprehensively characterized?

Immunoprecipitation coupled with chemical crosslinking followed by LC-MS/MS analysis has successfully identified the DNAJC15 protein interaction network . This approach revealed 185 significantly enriched proteins, with 179 being mitochondrial - primarily localized to the matrix and inner membrane . Key interactions include components of the TIM23 translocase (PAM16, TIMM23, TIMM44), mitochondrial quality control factors (AFG3L2, YME1L), and respiratory chain components (particularly complex I subunits) . For targeted validation of specific interactions, co-immunoprecipitation followed by Western blotting remains the method of choice.

What methodologies best assess DNAJC15's impact on mitochondrial protein import?

SILAC (stable isotope labeling with amino acids in cell culture) combined with cellular fractionation and proteomics provides comprehensive data on protein import dynamics . This approach revealed that DNAJC15 depletion reduced the mitochondrial localization of 188 proteins, with an additional 75 proteins showing impaired translocation across the outer membrane . For functional assessment of respiratory chain activity influenced by DNAJC15-mediated import, oxygen consumption measurements in isolated mitochondria demonstrate specific defects in complex I and II activities in DNAJC15-depleted conditions . These approaches allow for quantitative assessment of how DNAJC15 influences the mitochondrial proteome and resulting bioenergetic functions.

How does DNAJC15 contribute to mitochondrial protein import specificity?

DNAJC15 exhibits selective regulation of protein import through its association with specific TIM23 translocase complexes. Proteomic analysis revealed that DNAJC15 depletion specifically impairs the import of proteins destined for the matrix and inner membrane, particularly those with classical mitochondrial targeting sequences . Genetically, DNAJC15 interacts with TIMM17A but not its homolog TIMM17B, suggesting it cooperates with TIMM17A-containing complexes for importing specific subsets of proteins . This specificity explains why DNAJC15 depletion disproportionately affects oxidative phosphorylation components and mitochondrial gene expression machinery .

What is the functional relationship between DNAJC15 and its paralog DNAJC19?

DNAJC15 and DNAJC19 show significant functional redundancy in mitochondrial protein import. Both proteins are highly homologous in their J-domain and transmembrane regions but differ in their N-terminal structure . While single depletion of either protein has moderate effects, simultaneous depletion of DNAJC19 in DNAJC15-/- cells abolishes cell proliferation . Re-expression experiments demonstrated that DNAJC15 or DNAJC15D2 fully restores cell growth in double-depleted cells, while S-DNAJC15 (a specific variant) provides only partial recovery . Despite their redundancy, DNAJC15 has specific functions that cannot be fully compensated by DNAJC19, as evidenced by respiratory defects in DNAJC15-depleted mitochondria .

What happens to mitochondrial preproteins when DNAJC15 is absent?

When DNAJC15 is depleted, mitochondrial protein import is disrupted, leading to the accumulation of mitochondrial preproteins at the endoplasmic reticulum (ER) . This mislocalization disrupts ER proteostasis and triggers an ATF6-mediated unfolded protein response (UPR) . Interestingly, this does not activate the integrated stress response (ISR), the IRE1/XBP1-mediated UPR branch, oxidative stress response, or heat shock response . The selective activation of ATF6-mediated UPR highlights DNAJC15's role at the interface of mitochondrial and ER protein quality control systems.

How does DNAJC15 influence mitochondrial respiratory chain function?

DNAJC15 serves as a negative regulator of the mitochondrial respiratory chain under normal conditions, preventing mitochondrial hyperpolarization and restricting ATP generation . Paradoxically, DNAJC15 depletion significantly impairs complex I activity in both phosphorylation-coupled and uncoupled states, with moderate reductions in complex II activity . Oxygen consumption measurements in isolated mitochondria from DNAJC15-depleted cells show specific defects in complex I-dependent respiration . This apparent contradiction is resolved by understanding that DNAJC15 regulates the import of proteins essential for respiratory chain assembly and function, explaining why its absence leads to respiratory deficiency despite its role as a negative regulator .

What is the impact of DNAJC15 on cellular metabolism, particularly in cancer cells?

DNAJC15 significantly influences cellular metabolism, particularly lipid metabolism in cancer cells. When overexpressed, DNAJC15 promotes accumulation of lipid droplets and increases lipid peroxidation, creating conditions favorable for ferroptosis induction . These metabolic changes are linked to DNAJC15's role in mitochondrial function, as alterations in respiratory chain activity and oxidative stress can influence lipid metabolism . In ovarian cancer cells, high DNAJC15 levels are associated with decreased tumorigenic features, suggesting that DNAJC15-mediated metabolic reprogramming may suppress cancer progression .

How does DNAJC15 contribute to ferroptosis susceptibility in cancer cells?

DNAJC15 regulates susceptibility to ferroptosis, a form of regulated cell death characterized by iron-dependent accumulation of lipid peroxides. When overexpressed in ovarian cancer cells, DNAJC15 induces a phenotype displaying increased lipid peroxidation and subsequent ferroptosis . This mechanism was validated by showing that treatment with Ferrostatin-1 (a ferroptosis inhibitor) decreased cells' vulnerability to ferroptosis and restored cisplatin resistance despite DNAJC15 overexpression . The connection between DNAJC15, lipid metabolism, and ferroptosis represents a novel aspect of DNAJC15 biology with important implications for understanding cancer cell death mechanisms.

How is DNAJC15 expression altered in cancer and what are the implications?

DNAJC15 expression is frequently downregulated in various cancer types, particularly in ovarian and breast cancers . This downregulation correlates with cancer progression and acquisition of chemoresistance, particularly to cisplatin in ovarian cancer . Mechanistically, loss of DNAJC15 reduces ferroptosis susceptibility, providing a survival advantage to cancer cells and contributing to drug resistance . These findings suggest that DNAJC15 expression status could serve as a potential prognostic marker during cancer progression, with low expression indicating more aggressive disease and potential treatment resistance.

What therapeutic strategies might exploit DNAJC15 biology in cancer treatment?

Several therapeutic approaches emerge from our understanding of DNAJC15 in cancer:

• Ferroptosis modulation: In tumors with low DNAJC15 expression, combining cisplatin with ferroptosis inducers might overcome chemoresistance .

• Metabolic targeting: Therapies exploiting altered lipid metabolism associated with DNAJC15 loss could provide selective approaches for treating DNAJC15-deficient tumors .

• Synthetic lethality: The functional redundancy between DNAJC15 and DNAJC19 suggests that targeting DNAJC19 in DNAJC15-deficient tumors might create a synthetic lethal interaction .

• Mitochondrial stress sensitization: Since DNAJC15 loss affects mitochondrial protein import, compounds that further stress this pathway might selectively affect DNAJC15-deficient cancer cells .

How does the DNAJC15 interactome inform its multifunctional roles?

Proteomic analysis of DNAJC15 interactions revealed connections to multiple mitochondrial processes beyond protein import . Strong interactions with quality control factors (AFG3L2, YME1L, CLPB) and biogenesis factors (PHB1, PHB2, STOML2) suggest a role in coordinating protein import with quality control . Notably, DNAJC15 shows enriched interactions with enzymes involved in coenzyme Q metabolism and components of respiratory complex I assembly, particularly in the Q module and ND4 module . This interaction network positions DNAJC15 as a hub connecting protein import, quality control, and respiratory chain assembly, explaining its diverse cellular effects.

What is the significance of OMA1-mediated processing of DNAJC15?

The N-terminal region of DNAJC15 extending into the intermembrane space can be cleaved by the stress-activated protease OMA1 . This processing event likely represents a regulatory mechanism modulating DNAJC15 function under different conditions. Research suggests that the processed form (DNAJC15D2) maintains full functionality in supporting cell growth when reintroduced into DNAJC15-deficient cells, while a non-cleavable form (S-DNAJC15) provides only partial restoration . These observations indicate OMA1-mediated processing might represent an adaptive response to mitochondrial stress that alters DNAJC15's activity or interactions with other proteins.

How does DNAJC15 contribute to mitochondria-ER communication?

DNAJC15 depletion leads to accumulation of mitochondrial preproteins at the endoplasmic reticulum, disrupting ER proteostasis and triggering an ATF6-mediated unfolded protein response . This observation places DNAJC15 at the interface of mitochondria-ER communication. The mechanism behind this mistrafficking phenomenon represents a novel form of organelle crosstalk, where dysfunction in mitochondrial protein import directly impacts ER function. Understanding this relationship could provide insights into diseases involving mitochondrial-ER miscommunication, such as certain neurodegenerative disorders and metabolic diseases.

What are the tissue-specific functions of DNAJC15?

DNAJC15 shows tissue-specific expression patterns, with particularly high abundance in heart tissue in mice . This differential expression suggests potential tissue-specific functions that may reflect varying demands for mitochondrial protein import or respiratory chain regulation. The physiological implications of these expression patterns remain largely unexplored but could include specialized roles in tissues with high energetic demands. This represents an important area for future research, potentially using tissue-specific knockout models to dissect these functions while avoiding potential developmental defects associated with complete loss of DNAJC15.

How might DNAJC15 function be integrated into broader cellular adaptation mechanisms?

DNAJC15's position at the nexus of mitochondrial protein import, respiratory chain function, and stress responses suggests a potential role in coordinating cellular adaptation to changing environments. The selective activation of the ATF6-mediated UPR following DNAJC15 depletion, without triggering other stress pathways, indicates specificity in these adaptive mechanisms . Understanding how DNAJC15 function is regulated under different conditions (metabolic stress, hypoxia, nutrient limitation) could provide insights into cellular adaptation strategies. This integrated perspective represents an emerging frontier in DNAJC15 research with implications for understanding both normal physiology and disease states.