Turboid-based proximity labeling reveals SIRT5 network regulation in Acute Myeloid Leukemia cell lines Free

Background The SIRT5 lysine deacylase is a critical regulator of cellular metabolic homeostasis. We and others have shown that a large subset of acute myeloid leukemia (AML) samples is dependent on SIRT5 to maintain balanced metabolism to meet the needs of rapid cell proliferation. SIRT5-regulated pathways include glutaminolysis, redox homeostasis and oxidative phosphorylation, but the precise mechanisms by which SIRT5 regulates these metabolic functions are unknown. As SIRT5 dependence in AML is not correlated with any specific genotype, we hypothesized that SIRT5 dependence reflects a metabolic phenotype primarily encoded by SIRT5 interacting proteins. Taking advantage of the observation that certain AML cell lines exhibit vastly different levels of SIRT5 dependence, we mapped SIRT5 protein interaction networks across dependent and independent cell lines using proximity labeling to identify differences correlating with SIRT5 dependence and -independence.

Objective To map the SIRT5 interactome in SIRT5-dependent and -independent AML cell lines to establish correlations with phenotype.

Methods We selected AML cell lines (N=4) based on high level dependence or independence based on their response to SIRT5 knockdown.Cell lines were engineered to stably SIRT5 fused with biotin ligase (TurboID), starved of biotin for 72hrs before being incubated with 500 uM of biotin for 70 minutes. Cells were lysed and biotinylated proteins were pulled down using streptavidin conjugated beads followed by label-free mass spectrometry. The intensity values obtained for each detected protein were normalized, and the fold change was subsequently calculated by comparing the average expression between the replicates incubated with biotin and those without biotin. Proteins with a Log2FC > 1.5 were selected and those unique to each group (SIRT5-dependent and independent) were identified. The biological and molecular function of the protein groups obtained under each condition was determined using the UniProt, STRING, and DAVID databases. Based on the function and expression profiles of proteins in the SIRT5-dependent cell lines, selected candidates were chosen for validation by assessing their interaction through immunoprecipitation assays. SIRT5 was immunoprecipitated using streptavidin beads to confirm the interaction.

Results A total of 163 enriched proteins were identified in OCI-AML3 cells and 164 in KG1a cells (SIRT5-independent), and 176 and 199 in OCI-AML2 and SKM1 cells (SIRT5-dependent), respectively. Total of 327 proteins were enriched in the SIRT5-independent cell lines and 375 in the SIRT5-dependent lines. Eighty-three proteins were found in common between SIRT5-dependent and -independent cell lines and excluded from further analysis.

Functional enrichment analysis revealed that proteins from the SIRT5-independent lines are associated with distinct cellular processes, including ATP binding, nucleotide binding, and mitochondrial function —within this last category, 40 mitochondria-related proteins were identified. In contrast, the SIRT5-dependent group showed distinct annotations involving mitochondrial activity, mitochondrial matrix, and mitochondrial inner membrane, with 57 proteins falling under this category.

Interestingly, none of the 40 mitochondria-associated proteins in the independent group overlapped with the 57 found in the dependent group, suggesting that SIRT5 interactomes are distinct between SIRT5-dependent and -independent cell lines. DNAJC11 and TMEM126A were selected for downstream analysis based on co-localization with SIRT5 and their central role for mitochondrial processes, including cristae organization and Complex I assembly. Functional characterization of these candidates is underway and will be reported.

Conclusions Our findings reveal distinct differences between the SIRT5 interactomes of SIRT5-dependent and independent cell lines that may be causally related to their differential requirement for SIRT5 activity. Proteins involved in mitochondrial translation, electron transport, mitochondrial organization, and general translation processes may define the state of SIRT5 dependence and represent candidates for therapeutic intervention, alone or in conjunction with targeting SIRT5 activity.