DLST Human

What is DLST and what is its primary function in human cells?

DLST is the E2 component of the α-ketoglutarate (αKG) dehydrogenase complex, a critical enzyme complex in the tricarboxylic acid (TCA) cycle. Its primary function is to catalyze the conversion of α-ketoglutarate to succinyl-CoA in an irreversible oxidative decarboxylation reaction . This reaction is vital for the entry of glutamine into the TCA cycle and produces NADH for oxidative phosphorylation (OXPHOS) . DLST contains a catalytic domain responsible for generating succinyl-CoA, which serves as both a TCA cycle intermediate and a substrate for protein post-translational modifications .

Where is DLST primarily localized in human cells?

While DLST is predominantly active in the mitochondria as part of the 2-oxoglutarate dehydrogenase complex, research has revealed that a fraction of the complex also localizes to the nucleus . In the nucleus, DLST has been found to associate with KAT2A on chromatin and provides succinyl-CoA for histone succinyltransferase activity . This dual localization indicates that DLST functions in both metabolic processes and gene regulation through histone modifications .

How does DLST interact with other components of the 2-oxoglutarate dehydrogenase complex?

DLST works in concert with other components of the 2-oxoglutarate dehydrogenase complex (OGDHc), including oxoglutarate dehydrogenase (OGDH) and dihydrolipoamide dehydrogenase (DLD) . These three enzymes coordinate sequential reactions to convert α-ketoglutarate to succinyl-CoA with the concomitant reduction of NAD+ to NADH. DLST specifically handles the intermediate step where the succinyl group is transferred to coenzyme A . Research focusing on these protein-protein interactions often employs co-immunoprecipitation assays and structural biology techniques to elucidate the assembly and regulation of this multienzyme complex.

How do DLST mutations contribute to the development of pheochromocytomas and paragangliomas (PPGLs)?

Recurrent germline DLST mutations have been identified in individuals with multiple pheochromocytomas and paragangliomas (PPGLs) . These mutations, particularly those affecting the catalytic domain of DLST, disrupt normal enzyme function and alter cellular metabolism . The molecular mechanisms underlying PPGL development in patients with DLST mutations involve protein hyposuccinylation and metabolic remodeling . Specifically, DLST mutations lead to reduced production of succinyl-CoA, which affects post-translational modifications of proteins and alters global gene expression patterns through changes in histone succinylation .

What metabolic alterations occur in cancer cells with dysregulated DLST expression?

In cancer cells with dysregulated DLST expression, several key metabolic alterations have been observed:

• Altered glutamine metabolism and TCA cycle function due to changes in α-ketoglutarate conversion to succinyl-CoA

• Impaired NADH production affecting OXPHOS capacity

• Changes in cellular energy production pathways, forcing cancer cells to adapt their metabolic dependencies

• Alterations in protein succinylation patterns, affecting various cellular processes including gene expression through histone modifications

In neuroblastoma specifically, DLST depletion suppresses NADH production and impairs OXPHOS, leading to growth arrest and apoptosis of tumor cells, highlighting the dependency of these cancer cells on DLST-mediated metabolism .

What animal models are effective for studying DLST function in cancer progression?

Zebrafish models have proven particularly effective for studying DLST's role in cancer progression, especially in neuroblastoma. Research has employed MYCN-driven neuroblastoma zebrafish models (MYCN_TT) where human MYCN is expressed under the control of zebrafish dβh promoter . By introducing human DLST into neural crest cells through microinjection of Tg(dβh:DLST) constructs, researchers demonstrated that even modest increases (approximately 2-fold) in DLST expression collaborate with MYCN to promote neuroblastoma aggression . Conversely, monoallelic dlst loss was shown to impede disease initiation and progression . These models allow for temporal monitoring of tumor development, assessment of metastatic spread, and evaluation of therapeutic interventions in a physiologically relevant in vivo context.

What are the recommended methods for measuring DLST enzymatic activity in human tissue samples?

Measuring DLST enzymatic activity in human tissue samples typically involves spectrophotometric assays that monitor the rate of NAD+ reduction to NADH during the conversion of α-ketoglutarate to succinyl-CoA. The activity can be measured in isolated mitochondria or tissue homogenates, with careful consideration of sample preparation to preserve enzyme function. Advanced approaches may include:

• Isotope tracing experiments using 13C-labeled substrates followed by mass spectrometry to track metabolic flux through the DLST reaction

• In-gel activity assays following native PAGE separation of protein complexes

• Immunocapture-based activity measurements using DLST-specific antibodies

When analyzing clinical samples, it's crucial to establish appropriate normalization methods (e.g., relative to mitochondrial content or other housekeeping enzymes) and include controls that account for tissue-specific differences in basal metabolic activities.

What techniques are most effective for studying protein succinylation changes related to DLST function?

To study protein succinylation changes related to DLST function, researchers employ several complementary approaches:

• Immunoblotting with anti-succinyl-lysine antibodies for global assessment of protein succinylation levels

• Mass spectrometry-based proteomics, particularly using stable isotope labeling by amino acids in cell culture (SILAC) or tandem mass tag (TMT) labeling, to identify specific succinylation sites and quantify changes across conditions

• Enrichment of succinylated peptides using anti-succinyl-lysine antibodies before mass spectrometry analysis

• Chromatin immunoprecipitation (ChIP) assays to assess histone succinylation patterns at specific genomic loci

For comprehensive analysis, these approaches should be combined with functional studies that manipulate DLST levels (e.g., through RNA interference or CRISPR-Cas9 gene editing) to establish causal relationships between DLST activity and observed succinylation patterns .

How does nuclear DLST contribute to epigenetic regulation through histone succinylation?

Nuclear DLST has been identified as a key provider of succinyl-CoA for histone modifications. Research indicates that a fraction of the 2-oxoglutarate dehydrogenase complex localizes to the nucleus where DLST associates with KAT2A on chromatin . In this nuclear context, DLST provides the succinyl-CoA substrate required for histone succinyltransferase activity mediated by KAT2A . This represents a direct link between metabolism and gene regulation, as nuclear DLST activity can influence gene expression patterns through modulating histone post-translational modifications.

What is the mechanistic relationship between DLST and OXPHOS in high-risk neuroblastoma?

DLST plays a critical role in supporting oxidative phosphorylation (OXPHOS) in high-risk neuroblastoma through its involvement in NADH production. Studies have revealed that DLST depletion in human MYCN-amplified neuroblastoma cells significantly suppresses NADH production and impairs OXPHOS, leading to growth arrest and apoptosis . Interestingly, this DLST dependency appears to be somewhat specific to the OXPHOS pathway, as DLST depletion minimally affected glutamine anaplerosis and did not alter TCA cycle metabolites other than α-ketoglutarate .

The dependency on OXPHOS suggests a therapeutic vulnerability, as multiple inhibitors targeting the electron transport chain, including IACS-010759, have demonstrated efficacy in reducing neuroblastoma proliferation in vitro and suppressing tumor growth in zebrafish and mouse xenograft models . This represents a potential therapeutic approach for high-risk neuroblastoma patients, particularly those with elevated DLST expression.

How do post-translational modifications regulate DLST activity and stability?

While the role of DLST in mediating post-translational modifications (particularly succinylation) of other proteins is increasingly understood, less is known about how post-translational modifications regulate DLST itself. Research should address:

• The types and sites of post-translational modifications on DLST protein

• How these modifications affect DLST enzymatic activity, stability, and subcellular localization

• The enzymes responsible for adding or removing these modifications

• How modification patterns change in response to metabolic states or in disease conditions

Methodologically, this would involve mass spectrometry-based proteomics to map modification sites, site-directed mutagenesis to create modification-resistant variants, and in vitro enzymatic assays to assess functional consequences of specific modifications.

What strategies exist for therapeutically targeting DLST or its downstream pathways in cancer?

Based on the role of DLST in supporting cancer cell metabolism, several therapeutic strategies can be considered:

• Direct inhibition of DLST enzymatic activity through small molecule inhibitors, though this approach faces challenges related to specificity and potential metabolic compensation

• Targeting the electron transport chain and OXPHOS pathway, as demonstrated by the effectiveness of IACS-010759 (a potent inhibitor of ETC complex I) in suppressing neuroblastoma growth in vitro and in vivo models

• Combination approaches that simultaneously target DLST-dependent pathways and alternative metabolic routes that may serve as escape mechanisms

• Exploring synthetic lethality approaches by identifying genes that, when inhibited along with DLST, cause selective cancer cell death

The development of such therapies requires careful consideration of tissue-specific metabolic dependencies and potential toxicities to normal cells that rely on TCA cycle function.

How can DLST expression or mutation status be leveraged for patient stratification in precision oncology?

DLST expression or mutation status presents valuable opportunities for patient stratification in precision oncology:

• High DLST expression predicts poor survival and disease aggression in neuroblastoma patients, suggesting its potential as a prognostic biomarker

• DLST expression could identify patients likely to respond to OXPHOS inhibitors, as DLST-high tumors appear particularly dependent on this metabolic pathway

• Recurrent germline DLST mutations in pheochromocytoma and paraganglioma patients could inform genetic screening protocols for high-risk individuals

To effectively implement DLST-based stratification, standardized assays for measuring DLST expression or detecting mutations would need to be developed and validated in clinical laboratory settings. This would involve immunohistochemistry protocols with quantifiable scoring systems or targeted sequencing panels that include the DLST gene along with other known cancer-associated genes.

What biomarkers could serve as indicators of DLST activity or response to DLST-targeted therapies?

Several potential biomarkers could indicate DLST activity or response to DLST-targeted therapies:

• Metabolic biomarkers: Levels of TCA cycle intermediates, particularly the α-ketoglutarate to succinyl-CoA ratio, which directly reflects DLST activity

• Energetic biomarkers: NADH/NAD+ ratio and ATP production rates as indicators of OXPHOS function downstream of DLST

• Protein succinylation patterns: Global or specific protein succinylation levels as indicators of succinyl-CoA availability

• Imaging biomarkers: FDG-PET or other metabolic imaging approaches that can assess changes in tumor metabolism following therapy

Methodologically, these biomarkers could be assessed through mass spectrometry-based metabolomics, enzyme activity assays, immunohistochemistry for succinylated proteins, or functional imaging. Validation studies would need to establish clear thresholds for defining "high" versus "low" DLST activity and correlate these with clinical outcomes or response to specific therapies.