Biology subjects

Nguyen, T. V. H.

Publications and source records attributed to Nguyen, T. V. H..

3 recordsLinked to original sources

bioRxiv2026

Redox-dependent dimerization of PolDIP2 and a conserved ApaG-domain motif required for CHCHD2 interaction

PolDIP2 is a multifunctional mitochondrial protein implicated in redox regulation, mitochondrial proteostasis, and diverse mtDNA-associated processes, yet the principles underlying its regulation remain unclear. Crystallographic analysis revealed that PolDIP2 forms a redox-dependent disulfide-linked homodimer via a conserved Cys143 residue within its N-terminal YccV-like domain, and cellular and in vitro assays confirmed that this residue is essential for dimer formation. Oxidative stress enhanced dimerization of endogenous and ectopically expressed PolDIP2, and dimers were detected exclusively within mitochondria, requiring proper mitochondrial import. WT and C143A PolDIP2 overexpression produced similarly modest effects on mtDNA replication in cells, suggesting that dimerization has limited impact on mtDNA-associated processes. Proteomic analysis and biochemical validation identified both previously known and not yet characterized mitochondrial interactors of PolDIP2, and highlighted CHCHD2 as a specific binding partner. A conserved glycine-rich motif in the C-terminal ApaG/DUF525-like domain proved essential for this interaction, and disruption of the motif enhanced Cys143-dependent dimerization while abolishing CHCHD2 association, which preferentially occurs with monomeric PolDIP2. These findings define redox-controlled dimerization and a conserved ApaG-domain motif as key structural features shaping PolDIP2s interaction state within mitochondria and provide a basis for exploring its roles in redox-sensitive mitochondrial pathways.

cell biology↗

bioRxiv2024

The Y951N patient mutation inactivates the intramolecular switch in human mitochondrial DNA POLgamma

Mitochondrial DNA (mtDNA) stability, essential for cellular energy production, relies on DNA polymerase gamma (POL{gamma}). Here, we show that the POL{gamma} Y951N disease causing mutation induces replication stalling and severe mtDNA depletion. However, unlike other POL{gamma} disease causing mutations, Y951N does not directly impair exonuclease activity and only mildly affects polymerase activity. Instead, we found that Y951N compromises the enzymes ability to efficiently toggle between DNA synthesis and degradation, and is thus the first patient-derived mutation with impaired polymerase-exonuclease switching. These findings provide new insights into the intramolecular switch when POL{gamma} proofreads the newly-synthesized DNA strand, and reveal a new mechanism for causing mitochondrial DNA instability. Significance StatementDNA polymerase gamma (POL{gamma}) is essential for copying mitochondrial DNA (mtDNA), which is crucial for our energy production. POL{gamma} must accurately switch between making new DNA (polymerase activity) and correcting errors (exonuclease activity). While it is known that mutations in POL{gamma} can cause mitochondrial diseases by directly impairing these enzymatic functions, this study reveals a new mechanism. The Y951N mutation disrupts POL{gamma}s ability to switch between these activities, leading to severe blockages in DNA replication and a loss of mtDNA in human cells, even without significant direct impairment of polymerase or exonuclease activities. These findings provide new insights into the origins of mitochondrial diseases.

biochemistry↗

bioRxiv2024

The specific AMPK activator A-769662 ameliorates pathological phenotypes following mitochondrial DNA depletion

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis that also plays a role in preserving mitochondrial function and integrity. Upon a disturbance in the cellular energy state that increases AMP levels, AMPK activity promotes a switch from anabolic to catabolic metabolism to restore energy homeostasis. However, it is currently unclear how severe of a mitochondrial dysfunction is required to trigger AMPK activation, and whether stimulation of AMPK using specific agonists can improve the cellular phenotype following mitochondrial dysfunction. Using a cell model of mitochondrial disease characterized by progressive mitochondrial DNA (mtDNA) depletion and deteriorating mitochondrial metabolism, we show that mitochondria-associated AMPK becomes activated early in the course of the advancing mitochondrial dysfunction, before any quantifiable decrease in the ATP/(AMP+ADP) ratio or respiratory chain activity. Moreover, stimulation of AMPK activity using the specific small-molecule agonist A-769662 alleviated the mitochondrial phenotypes caused by the mtDNA depletion and restored normal mitochondrial membrane potential. Notably, the agonist treatment was able to partially restore mtDNA levels in cells with severe mtDNA depletion, while it had no impact on mtDNA levels of control cells. The beneficial impact of the agonist was also observed in cells from patients suffering from mtDNA depletion. However, the positive effects of A-769662 in the two experimental cell models appeared to involve at least partially different mechanisms. These findings improve our understanding of the effects of specific small-molecule activators of AMPK on mitochondrial and cellular function, and suggest a potential utility for these compounds in disease states involving mtDNA depletion.

molecular biology↗