Regulation Of Protein Function Research Articles

Phospholipid Membranes as Chemically and Functionally Tunable Materials.

The sheet-like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to the formation of a bilayer in aqueous environment. Lipids are not just structural elements. Individual lipid species, the lipid membrane structure, and lipid dynamics influence and regulate membrane protein function. An exciting field is emerging where the membrane-associated material properties of different bilayer systems are used in designing innovative solutions for widespread applications across various fields, such as the food industry, cosmetics, nano- and biomedicine, drug storage and delivery, biotechnology, nano- and biosensors, and computing. Here, the authors summarize what is known about how lipids determine the properties and functions of biological membranes and how this has been or can be translated into innovative applications. Based on recent progress in the understanding of membrane structure, dynamics, and physical properties, a perspective is provided on how membrane-controlled regulation of protein functions can extend current applications and even offer new applications.

SENP3 mediates the activation of the Wnt/β-catenin signaling pathway to accelerate the growth and metastasis of oesophagal squamous cell carcinoma in mice.

As a common malignant tumor, esophageal squamous cell carcinoma (ESCC) is occasionally seen in clinical practice. This type of disease has low incidence rate and mortality. The post-translational modification of small ubiquitin like modifiers (SUMO) can play a crucial role in regulating protein function, and can significantly impact the occurrence and development of diseases. SUMO-specific peptidase (SENP) affects cell activity by regulating the biological function of SUMO. SENP3 belongs to the SENP family, and available data indicate that many malignancies are associated with SENPs, it is currently unclear its role in ESCC. This study indicates that there is a high level of SENP3 expression in ESCC tumor cells. If the expression level of this gene is high, it can have a significant impact on ESCC cell lines and affect physiological activities such as invasion of KYSE170 cells. If the gene is knocked out, this situation will not occur. There is also research data indicating that this gene can effectively activate related signaling pathways, thereby promoting the physiological activities of malignant tumor cells. In a nude mouse xenograft tumor model, KYSE170 cells with SENP3 expression knockdown induced a smaller volume and weight of tumor tissue. Therefore, it can be clearly stated that SENP3 can enable Wnt/ β- The catenin signaling pathway is stimulated, which in turn affects the physiological activities of ESCC cells, including the invasion process. The results of this article lay the foundation for clinical staff to carry out clinical management.

An elastase nanocomplex with metal cofactors for enhancement of target protein cleavage activity and synergistic antitumor effect

Neutrophil elastase (ELANE) plays a vital role in recognizing and destroying various cancer cells by cleaving target proteins, making it a promising therapeutic option. However, in vivo therapeutic effectiveness of ELANE is limited due to its inadequate protein cleavage efficiency, lack of tumor tissue targeting, and susceptibility to serine protease inhibition. Drawing inspiration from the ways metal ions regulate protein functions and activities, a metal cofactor-based approach was employed to enhance target protein cleavage and antitumor effects, using porcine pancreatic elastase (PPE, an ELANE homolog) as a model drug in this study. In a nutshell, an elastase nanocomplex with enhanced cleavage activity (CAEN) was developed and optimized in vitro. Administered through inhalation, CAEN prevented the degradation of PPE in the biological environment and efficiently delivered it to tumor cells. By effectively cleaving the transmembrane protein CD95 with higher specificity compared to free PPE, CAEN induced significant apoptosis in lung cancer cells. Furthermore, the released metal ions could activate type I interferon in macrophages, further enhancing the local immune response within lung tumor tissues. Overall, this study presents a novel approach to substantially enhance the specificity of protein drugs in terms of drug delivery and pharmacological effects.

Genome-wide identification of U-box gene family and expression analysis in response to saline-alkali stress in foxtail millet (Setaria italica L. Beauv).

E3 ubiquitin ligases are central modifiers of plant signaling pathways that regulate protein function, localization, degradation, and other biological processes by linking ubiquitin to target proteins. E3 ubiquitin ligases include proteins with the U-box domain. However, there has been no report about the foxtail millet (Setaria italica L. Beauv) U-box gene family (SiPUB) to date. To explore the function of SiPUBs, this study performed genome-wide identification of SiPUBs and expression analysis of them in response to saline-alkali stress. A total of 70 SiPUBs were identified, which were unevenly distributed on eight chromosomes. Phylogenetic and conserved motif analysis demonstrated that SiPUBs could be clustered into six subfamilies (I-VI), and most SiPUBs were closely related to the homologues in rice. Twenty-eight types of cis-acting elements were identified in SiPUBs, most of which contained many light-responsive elements and plant hormone-responsive elements. Foxtail millet had 19, 78, 85, 18, and 89 collinear U-box gene pairs with Arabidopsis, rice, sorghum, tomato, and maize, respectively. Tissue specific expression analysis revealed great variations in SiPUB expression among different tissues, and most SiPUBs were relatively highly expressed in roots, indicating that SiPUBs may play important roles in root development or other growth and development processes of foxtail millet. Furthermore, the responses of 15 SiPUBs to saline-alkali stress were detected by qRT-PCR. The results showed that saline-alkali stress led to significantly differential expression of these 15 SiPUBs, and SiPUB20/48/70 may play important roles in the response mechanism against saline-alkali stress. Overall, this study provides important information for further exploration of the biological function of U-box genes.

Profiling of deubiquitinases that control virulence in the pathogenic plant fungus Fusarium graminearum.

Eukaryotes have evolved sophisticated post-translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin-conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown. Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world-wide. Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin-specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy-related protein Atg8, the mitogen-activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively. Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases.

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293 and HELA cells. But the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1, but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Cysteine thiol sulfinic acid in plant stress signaling.

Cysteine thiols are susceptible to various oxidative posttranslational modifications (PTMs) due to their high chemical reactivity. Thiol-based PTMs play a crucial role in regulating protein functions and are key contributors to cellular redox signaling. Although reversible thiol-based PTMs, such as disulfide bond formation, S-nitrosylation, and S-glutathionylation, have been extensively studied for their roles in redox regulation, thiol sulfinic acid (-SO2H) modification is often perceived as irreversible and of marginal significance in redox signaling. Here, we revisit this narrow perspective and shed light on the redox regulatory roles of -SO2H in plant stress signaling. We provide an overview of protein sulfinylation in plants, delving into the roles of hydrogen peroxide-mediated and plant cysteine oxidase-catalyzed formation of -SO2H, highlighting the involvement of -SO2H in specific regulatory signaling pathways. Additionally, we compile the existing knowledge of the -SO2H reducing enzyme, sulfiredoxin, offering insights into its molecular mechanisms and biological relevance. We further summarize current proteomic techniques for detecting -SO2H and furnish a list of experimentally validated cysteine -SO2H sites across various species, discussing their functional consequences. This review aims to spark new insights and discussions that lead to further investigations into the functional significance of protein -SO2H-based redox signaling in plants.

Amino acid and protein specificity of protein fatty acylation in Caenorhabditis elegans

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S-acyl moieties differ from N- and O-fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S-acylated proteins and 510 hydroxylamine-resistant N- or O-acylated proteins. Subsets of S-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

Designed allosteric protein logic.

The regulation of protein function by external or internal signals is one of the key features of living organisms. The ability to directly control the function of a selected protein would represent a valuable tool for regulating biological processes. Here, we present a generally applicable regulation of proteins called INSRTR, based on inserting a peptide into a loop of a target protein that retains its function. We demonstrate the versatility and robustness of coiled-coil-mediated regulation, which enables designs for either inactivation or activation of selected protein functions, and implementation of two-input logic functions with rapid response in mammalian cells. The selection of insertion positions in tested proteins was facilitated by using a predictive machine learning model. We showcase the robustness of the INSRTR strategy on proteins with diverse folds and biological functions, including enzymes, signaling mediators, DNA binders, transcriptional regulators, reporters, and antibody domains implemented as chimeric antigen receptors in T cells. Our findings highlight the potential of INSRTR as a powerful tool for precise control of protein function, advancing our understanding of biological processes and developing biotechnological and therapeutic interventions.

OXCT1 functions as a succinyltransferase, contributing to hepatocellular carcinoma via succinylating LACTB

Metabolic reprogramming is an important feature of cancers that has been closely linked to post-translational protein modification (PTM). Lysine succinylation is a recently identified PTM involved in regulating protein functions, whereas its regulatory mechanism and possible roles in tumor progression remain unclear. Here, we show that OXCT1, an enzyme catalyzing ketone body oxidation, functions as a lysine succinyltransferase to contribute to tumor progression. Mechanistically, we find that OXCT1 functions as a succinyltransferase, with residue G424 essential for this activity. We also identified serine beta-lactamase-like protein (LACTB) as a main target of OXCT1-mediated succinylation. Extensive succinylation of LACTB K284 inhibits its proteolytic activity, resulting in increased mitochondrial membrane potential and respiration, ultimately leading to hepatocellular carcinoma (HCC) progression. In summary, this study establishes lysine succinyltransferase function of OXCT1 and highlights a link between HCC prognosis and LACTB K284 succinylation, suggesting a potentially valuable biomarker and therapeutic target for further development.

Charge block-driven liquid-liquid phase separation: A mechanism of how phosphorylation regulates phase behavior of disordered proteins.

Phosphorylation regulates protein function by modulating stereospecific interactions between protein-protein or enzyme-ligand. On the other hand, many bioinformatics studies have demonstrated that phosphorylation preferably occurs in intrinsically disordered regions (IDRs), which do not have any secondary and tertiary structures. Although studies have demonstrated that phosphorylation changes the phase behavior of IDRs, the mechanism, which is distinct from the "stereospecific" effect, had not been elucidated. Here, we describe how phosphorylation in IDRs regulates the protein function by modulating phase behavior. Mitotic phosphorylation in the IDRs of Ki-67 and NPM1 promotes or suppresses liquid-liquid phase separation, respectively, by altering the "charge blockiness" along the polypeptide chain. The phosphorylation-mediated regulation of liquid-liquid phase separation by enhancing or suppressing "charge blockiness," rather than by modulating stereospecific interactions, may provide one of the general mechanisms of protein regulation by posttranslational modifications and the role of multiple phosphorylations.

Targeting the ubiquitin-proteasome system: a novel therapeutic strategy for neuroblastoma.

Neuroblastoma (NB) stands as a common and formidable malignant tumor among children, characterized by marked tumor heterogeneity and resistance to conventional treatments. Central to the regulation of protein stability, localization, and function is the process of ubiquitination-a critical protein modification. The therapeutic potential of drugs that target deubiquitination, demonstrated in the treatment of refractory multiple myeloma, warrants investigation in the context of NB. This review endeavors to demystify the intricate biological implications of ubiquitination within NB pathology, synthesize the current landscape of preclinical studies focused on the inhibition of the ubiquitin-proteasome system in NB, and assess the viability of this strategy as an innovative therapeutic frontier.

Analysis of protein-protein and protein-membrane interactions by isotope-edited infrared spectroscopy.

The objective of this work is to highlight the power of isotope-edited Fourier transform infrared (FTIR) spectroscopy in resolving important problems encountered in biochemistry, biophysics, and biomedical research, focusing on protein-protein and protein membrane interactions that play key roles in practically all life processes. An overview of the effects of isotope substitutions in (bio)molecules on spectral frequencies and intensities is given. Data are presented demonstrating how isotope-labeled proteins and/or lipids can be used to elucidate enzymatic mechanisms, the mode of membrane binding of peripheral proteins, regulation of membrane protein function, protein aggregation, and local and global structural changes in proteins during functional transitions. The use of polarized attenuated total reflection FTIR spectroscopy to identify the spatial orientation and the secondary structure of a membrane-bound interfacial enzyme and the mode of lipid hydrolysis is described. Methods of production of site-directed, segmental, and domain-specific labeling of proteins by the synthetic, semisynthetic, and recombinant strategies, including advanced protein engineering technologies such as nonsense suppression and frameshift quadruplet codons are overviewed.

METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5.

Background: Pathological cardiac hypertrophy, a condition that contributes to heart failure, is characterized by its intricate pathogenesis. The meticulous regulation of protein function, localization, and degradation is a crucial role played by deubiquitinating enzymes in cardiac pathophysiology. This study clarifies the participation and molecular mechanism of OTUD1 (OTU Deubiquitinase 1) in pathological cardiac hypertrophy. Methods: We generated a cardiac-specific Otud1 knockout mouse line (Otud1-CKO) and adeno-associated virus serotype 9-Otud1 mice to determine the role of Otud1 in cardiac hypertrophy. Its impact on cardiomyocytes enlargement was investigated using the adenovirus. RNA immunoprecipitation was used to validate the specific m6a methyltransferase interacted with OTUD1 transcript. RNA sequencing in conjunction with immunoprecipitation-mass spectrometry analysis was employed to identify the direct targets of OTUD1. A series of depletion mutant plasmids were constructed to detect the interaction domain of OTUD1 and its targets. Results: Ang II-stimulated neonatal rat cardiac myocytes and mice hearts subjected to transverse aortic constriction (TAC) showed increased protein levels of Otud1. Cardiac hypertrophy and dysfunction were less frequent in Otud1-CKO mice during TAC treatment, while Otud1 overexpression worsened cardiac hypertrophy and remodeling. METTL3 mediated m6A modification of OTUD1 transcript promoted mRNA stability and elevated protein expression. In terms of pathogenesis, Otud1 plays a crucial role in cardiac hypertrophy by targeting Pgam5, leading to the robust activation of the Ask1-p38/JNK signal pathway to accelerate cardiac hypertrophy. Significantly, the pro-hypertrophy effects of Otud1 overexpression were largely eliminated when Ask1 knockdown. Conclusion: Our findings confirm that targeting the OTUD1-PGAM5 axis holds significant potential as a therapeutic approach for heart failure associated with pathological hypertrophy.

Identification of Protein Targets of S-Nitroso-Coenzyme A-Mediated S-Nitrosation Using Chemoproteomics.

S-Nitrosation is a cysteine post-translational modification fundamental to cellular signaling. This modification regulates protein function in numerous biological processes in the nervous, cardiovascular, and immune systems. Small molecule or protein nitrosothiols act as mediators of NO signaling by transferring the NO group (formally NO+) to a free thiol on a target protein through a transnitrosation reaction. The protein targets of specific transnitrosating agents and the extent and functional effects of S-nitrosation on these target proteins have been poorly characterized. S-nitroso-coenzyme A (CoA-SNO) was recently identified as a mediator of endogenous S-nitrosation. Here, we identified direct protein targets of CoA-SNO-mediated transnitrosation using a competitive chemical-proteomic approach that quantified the extent of modification on 789 cysteine residues in response to CoA-SNO. A subset of cysteines displayed high susceptibility to modification by CoA-SNO, including previously uncharacterized sites of S-nitrosation. We further validated and functionally characterized the functional effects of S-nitrosation on the protein targets phosphofructokinase (platelet type), ATP citrate synthase, and ornithine aminotransferase.

Site-Specific Identification of Protein S-Acylation by IodoTMT0 Labeling and Immobilized Anti-TMT Antibody Resin Enrichment.

Protein S-acylation is a reversible post-translational modification (PTM). It is present on diverse proteins and has important roles in regulating protein function. Aminolysis with hydroxylamine is widely used in the global identification of the PTM. However, the identification is indirect. Distinct criteria have been used for identification, and the false discovery rate has not been addressed. Here, we report a site-specific method for S-acylation identification based on tagging of S-acylation sites with iodoTMT0. Efforts to improve the performance of the method and confidence of identification are discussed, highlighting the importance of reducing contaminant peptides and keeping the recovery rate consistent between aliquots with or without hydroxylamine treatment. With very stringent criteria, presumptive S-acylation sites of 269, 684, 695, and 780 were identified from HK2 cells, HK11 cells, mouse brain, and mouse liver samples, respectively. Among them, the newly identified protein S-acylation sites are equivalent to 34% of human and 24% of mouse S-acylation sites reported previously. In addition, false-positive rates for S-acylation identification and S-acylation abundances were estimated. Significant differences in S-acylation abundance were found from different samples (from 0.08% in HK2 cells to 0.76% in mouse brain), and the false-positive rates were significantly higher for samples with a low abundance of S-acylation.

High-throughput N-glycoproteomics with fast liquid chromatographic separation

N-glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N-glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N-glycosylation at the intact N-glycopeptide level, providing comprehensive information of peptide backbones, N-glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N-glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N-glycopeptides were identified, where 422 site- and structure-specific differential N-glycosylation on 145 N-glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N-glycoproteomes at large scale.

SNO-DCA: A model for predicting S-nitrosylation sites based on densely connected convolutional networks and attention mechanism

Protein S-nitrosylation is a reversible oxidative reduction post-translational modification that is widely present in the biological community. S-nitrosylation can regulate protein function and is closely associated with a variety of diseases, thus identifying S-nitrosylation sites are crucial for revealing the function of proteins and related drug discovery. Traditional experimental methods are time-consuming and expensive; therefore, it is necessary to explore more efficient computational methods. Deep learning algorithms perform well in the field of bioinformatics sites prediction, and many studies show that they outperform existing machine learning algorithms. In this work, we proposed a deep learning algorithm-based predictor SNO-DCA for distinguishing between S-nitrosylated and non-S-nitrosylated sequences. First, one-hot encoding of protein sequences was performed. Second, the dense convolutional blocks were used to capture feature information, and an attention module was added to weigh different features to improve the prediction ability of the model. The 10-fold cross-validation and independent testing experimental results show that our SNO-DCA model outperforms existing S-nitrosylation sites prediction models under imbalanced data. In this paper, a web server prediction website: https://sno.cangmang.xyz/SNO-DCA/was established to provide an online prediction service for users. SNO-DCA can be available at https://github.com/peanono/SNO-DCA.

The function of alternative splicing in the proteome: rewiring protein interactomes to put old functions into new contexts.

Alternative splicing affects more than 95% of multi-exon genes in the human genome. These changes affect the proteome in a myriad of ways. Here, we review our understanding of the breadth of these changes from their effect on protein structure to their influence on interactions. These changes encompass effects on nucleic acid binding in the nucleus to protein-carbohydrate interactions in the extracellular milieu, altering interactions involving all major classes of biological molecules. Protein isoforms have profound influences on cellular and tissue physiology, for example, by shaping neuronal connections, enhancing insulin secretion by pancreatic beta cells and allowing for alternative viral defense strategies in stem cells. More broadly, alternative splicing enables repurposing proteins from one context to another and thereby contributes to both the evolution of new traits as well as the creation of disease-specific interactomes that drive pathological phenotypes. In this Review, we highlight this universal character of alternative splicing as a central regulator of protein function with implications for almost every biological process.

Targeting Sumoylation Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by aberrant immature myeloid progenitor blasts in bone marrow and peripheral blood. Venetoclax (VEN), a BCL-2 inhibitor, is FDA-approved for AML treatment with hypomethylating agents (HMA). Despite the improvement of VEN and HMA, not all patients respond, and most patients develop resistance. This presents an urgent need for new therapies to overcome VEN resistance and enhance VEN efficacy. We propose SUMOylation inhibition as a novel therapy to address this need. SUMOylation regulates protein function by covalently attaching Small Ubiquitin-like MOdifier (SUMO) proteins to target proteins. SUMOylation plays an important role in tumor progression and immune response. Our study aims to evaluate the effects of SUMOylation inhibition on anti-AML activity of VEN and the mechanism. We firstly generated VEN-resistant cell lines Molm13-VR and MV411-VR by culturing Molm13 and MV4-11 cells in increasing doses of VEN. SUMO E1 (SAE2) and global SUMOylation levels were remarkably upregulated in VEN-resistant cell lines, suggesting SUMOylation plays a role in VEN resistance. We evaluated the antileukemic effects of TAK-981 (subasumstat), a novel and specific SUMO E1 inhibitor, alone and in combination with VEN, using AML cell lines and primary blasts isolated from refractory/relapsed (R/R) AML patients. Different AML cell lines and blast samples showed various sensitivities to VEN (IC 50 10nM ~ 5,000nM), but significant synergism was observed with TAK-981 and VEN in cell viability assay. TAK-981 synergized with VEN at inducing AML cell apoptosis determined by annexin-V staining with flow cytometry as well as increased levels of cleaved-PARP and cleaved caspase-3 by western blotting. The mechanism of action of VEN is dependent on BCL-2 mediated mitochondrial function and metabolism. We assessed mitochondrial membrane potential using TMRM probe. TAK-981 reduced mitochondrial membrane potential in both VEN-sensitive and resistant AML lines, further in combination with VEN. Because mitochondrial membrane potential is maintained by normal mitochondrial structure, we performed electron microscopy to determine mitochondrial structure. VEN had limited effect on mitochondria in resistant cells. However, TAK-981 induced abnormal ultrastructure mitochondria with fewer cristae, worsened by TAK-981+VEN combination. Since TAK-981 treatment caused mitochondrial structure defects, we determine the mitochondrial function using Seahorse XF assay. VEN decreased oxygen consumption rate (OCR), indicating inhibition of oxidative phosphorylation (OXPHOS), further suppressed by TAK-981. Metabolomic profiling by mass spectrometry indicated TAK-981+VEN treated AML cells displayed striking decreases in TCA cycle intermediates and reduced electron transport chain (ETC) activity. Pathway analysis of the metabolomics data revealed significant enrichment in amino acids metabolism. Notably, RNA-sequencing and GSEA analysis showed consistent findings that TAK-981+VEN showed suppressed signaling pathways in mitochondrial function, glycolysis and amino-acid metabolisms, indicating TAK-981 targeted mitochondrial metabolism to enhance VEN activity. We then determined whether TAK-981 enhanced antileukemic effect of VEN ex vivo and in vivo. TAK-981 synergized with VEN at reducing colony formation capacity in colony formation assay using leukemia stem cells (LSCs) isolated from relapse AML blasts. More importantly, TAK-981 showed remarkable therapeutic effect in patient-derived xenograft (PDX) mouse model engrafted with blasts isolated from R/R AML patients, and significantly synergized with VEN at extending mice survival in vivo. In conclusion, TAK-981 enhances the antileukemic activity of VEN by targeting mitochondria function and metabolism, offering a potent therapy for refractory/relapsed AML.