Assembly Of Protein Complexes Research Articles

COPII cage assembly factor Sec13 integrates information flow regulating endomembrane function in response to human variation

How information flow is coordinated for managing transit of 1/3 of the genome through endomembrane pathways by the coat complex II (COPII) system in response to human variation remains an enigma. By examining the interactome of the COPII cage-assembly component Sec13, we show that it is simultaneously associated with multiple protein complexes that facilitate different features of a continuous program of chromatin organization, transcription, translation, trafficking, and degradation steps that are differentially sensitive to Sec13 levels. For the trafficking step, and unlike other COPII components, reduction of Sec13 expression decreased the ubiquitination and degradation of wild-type (WT) and F508del variant cargo protein cystic fibrosis transmembrane conductance regulator (CFTR) leading to a striking increase in fold stability suggesting that the events differentiating export from degradation are critically dependent on COPII cage assembly at the ER Golgi intermediate compartment (ERGIC) associated recycling and degradation step linked to COPI exchange. Given Sec13’s multiple roles in protein complex assemblies that change in response to its expression, we suggest that Sec13 serves as an unanticipated master regulator coordinating information flow from the genome to the proteome to facilitate spatial covariant features initiating and maintaining design and function of membrane architecture in response to human variation.

Unconventional PDZ Recognition Revealed in α7 nAChR-PICK1 Complexes.

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

SUMO and the DNA damage response.

The preservation of genome integrity requires specialised DNA damage repair (DDR) signalling pathways to respond to each type of DNA damage. A key feature of DDR is the integration of numerous post-translational modification signals with DNA repair factors. These modifications influence DDR factor recruitment to damaged DNA, activity, protein-protein interactions, and ultimately eviction to enable access for subsequent repair factors or termination of DDR signalling. SUMO1-3 (small ubiquitin-like modifier 1-3) conjugation has gained much recent attention. The SUMO-modified proteome is enriched with DNA repair factors. Here we provide a snapshot of our current understanding of how SUMO signalling impacts the major DNA repair pathways in mammalian cells. We highlight repeating themes of SUMO signalling used throughout DNA repair pathways including the assembly of protein complexes, competition with ubiquitin to promote DDR factor stability and ubiquitin-dependent degradation or extraction of SUMOylated DDR factors. As SUMO 'addiction' in cancer cells is protective to genomic integrity, targeting components of the SUMO machinery to potentiate DNA damaging therapy or exacerbate existing DNA repair defects is a promising area of study.

Early insights into co-translational assembly of protein complexes.

Early insights into co-translational assembly of protein complexes.

Alteration of synaptic protein composition during developmental synapse maturation.

The postsynaptic density (PSD) is a collection of specialized proteins assembled beneath the postsynaptic membrane of dendritic spines. The PSD proteome comprises ~1000 proteins, including neurotransmitter receptors, scaffolding proteins and signalling enzymes. Many of these proteins have essential roles in synaptic function and plasticity. During brain development, changes are observed in synapse density and in the stability and shape of spines, reflecting the underlying molecular maturation of synapses. Synaptic protein composition changes in terms of protein abundance and the assembly of protein complexes, supercomplexes and the physical organization of the PSD. Here, we summarize the developmental alterations of postsynaptic protein composition during synapse maturation. We describe major PSD proteins involved in postsynaptic signalling that regulates synaptic plasticity and discuss the effect of altered expression of these proteins during development. We consider the abnormality of synaptic profiles and synaptic protein composition in the brain in neurodevelopmental disorders such as autism spectrum disorders. We also explain differences in synapse development between rodents and primates in terms of synaptic profiles and protein composition. Finally, we introduce recent findings related to synaptic diversity and nanoarchitecture and discuss their impact on future research. Synaptic protein composition can be considered a major determinant and marker of synapse maturation in normality and disease.

Diverging co-translational protein complex assembly pathways are governed by interface energy distribution

Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find that only a few residues serve as “hotspots,” initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes’ complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.

Abstract 6115: Genetic ancestry specific meQTLs control immune function regulation in a breast cancer cohort of African and European patients

Abstract Black women with significant African ancestry experience a disproportionately higher incidence of the most aggressive and invasive breast cancer subtypes, along with worse clinical outcomes across all demographic groups within the United States. These health disparities arise from complex interactions between genetic, sociocultural, and environmental factors. Epigenetic mechanisms, such as DNA methylation (DNAm), which regulate gene expression have been proposed as a means to capture the molecular impact of adverse environments on tumor biology. While DNAm levels can be influenced by environmental stimuli, they are also regulated by individual genotypes at methylation quantitative trait loci (meQTLs), posing a challenge to modeling the impact of the environment on cancer molecular characteristics. We hypothesized that ancestry differential DNAm in breast cancer is a result of differential genotype frequencies of SNPs due to ancestry at meQTL loci that can result in phenotypic differences between breast cancer patients of African and European ancestry through differential gene expression regulation. To investigate this hypothesis, we leveraged multi-omic data from The Cancer Genome Atlas (TCGA) breast cancer cohort to perform integrative analysis in patients of European and African ancestry (n=578). 804 CpGs were significantly differentially methylated between European and African ancestry breast cancer patients. meQTL analysis at these sites identified 37 cis-meQTLs and 748 trans-meQTLs, regulating methylation at CpG sites enriched in CpG Islands, enhancers, and other open chromatin regions. 54% of the cis- and 69% of the trans-meQTLs show evidence of differential genotype frequencies by ancestry. Association analysis between methylation and local gene expression revealed that 683 of the 804 ancestry differentially methylated sites regulate the expression of 5,774 genes. We found a statistically significant enrichment for genes involved in immune-related functions such as MHC class II protein complex assembly and the regulation of leukocyte cell-cell adhesion. Intriguingly, 10.5% of these genes encode transcription factors (TFs), and out of these ~23% are linked to African-ancestry specific open chromatin regions in breast cancer cell lines derived from African-ancestry donors. Our findings show that ancestry specific DNAm differences display strong genetic associations via ancestry associated meQTLs, affecting fundamental immunological processes in breast cancer. Our findings suggest that the effects of ancestry-specific genetic regulation may be more profound than previously appreciated, while pointing to the potential for confounding due to genetic ancestry when using DNA methylation as a molecular readout of environmental exposures. Citation Format: Kyriaki Founta, Nyasha Chambwe. Genetic ancestry specific meQTLs control immune function regulation in a breast cancer cohort of African and European patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6115.

Fungal heat shock proteins: molecular phylogenetic insights into the host takeover.

Heat shock proteins are constitutively expressed chaperones induced by cellular stress, such as changes in temperature, pH, and osmolarity. These proteins, present in all organisms, are highly conserved and are recruited for the assembly of protein complexes, transport, and compartmentalization of molecules. In fungi, these proteins are related to their adaptation to the environment, their evolutionary success in acquiring new hosts, and regulation of virulence and resistance factors. These characteristics are interesting for assessment of the host adaptability and ecological transitions, given the emergence of infections by these microorganisms. Based on phylogenetic inferences, we compared the sequences of HSP9, HSP12, HSP30, HSP40, HSP70, HSP90, and HSP110 to elucidate the evolutionary relationships of different fungal organisms to suggest evolutionary patterns employing the maximum likelihood method. By the different reconstructions, our inference supports the hypothesis that these classes of proteins are associated with pathogenic gains against endothermic hosts, as well as adaptations for phytopathogenic fungi.

Analytical Framework to Understand the Origins of Methyl Side-Chain Dynamics in Protein Assemblies.

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ1/χ2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to μs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

SUMO modifies GβL and mediates mTOR signaling

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL (G protein β-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GβL but not SUMOylation-defective KR mutant GβL promotes mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Assembly of Protein Complexes in and on the Membrane with Predicted Spatial Arrangement Constraints

Membrane proteins play crucial roles in various cellular processes, and their interactions with other proteins in and on the membrane are essential for their proper functioning. While an increasing number of structures of more membrane proteins are being determined, the available structure data is still sparse. To gain insights into the mechanisms of membrane protein complexes, computational docking methods are necessary due to the challenge of experimental determination. Here, we introduce Mem-LZerD, a rigid-body membrane docking algorithm designed to take advantage of modern membrane modeling and protein docking techniques to facilitate the docking of membrane protein complexes. Mem-LZerD is based on the LZerD protein docking algorithm, which has been constantly among the top servers in many rounds of CAPRI protein docking assessment. By employing a combination of geometric hashing, newly constrained by the predicted membrane height and tilt angle, and model scoring accounting for the energy of membrane insertion, we demonstrate the capability of Mem-LZerD to model diverse membrane protein–protein complexes. Mem-LZerD successfully performed unbound docking on 13 of 21 (61.9%) transmembrane complexes in an established benchmark, more than shown by previous approaches. It was additionally tested on new datasets of 44 transmembrane complexes and 92 peripheral membrane protein complexes, of which it successfully modeled 35 (79.5%) and 15 (16.3%) complexes respectively. When non-blind orientations of peripheral targets were included, the number of successes increased to 54 (58.7%). We further demonstrate that Mem-LZerD produces complex models which are suitable for molecular dynamics simulation. Mem-LZerD is made available at https://lzerd.kiharalab.org.

High-Throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF.

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil for human lungs. The pulmonary extracellular matrix is increasingly recognized as an independent bioactive entity, by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome ("matrisome") remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize the depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key core matrisome proteins.

Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

Computational analysis of protein synthesis, diffusion, and binding in compartmental biochips

Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the system’s three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.

OsALB3 Is Required for Chloroplast Development by Promoting the Accumulation of Light-Harvesting Chlorophyll-Binding Proteins in Rice

ALBINO3 (ALB3) protein functions in the insertion and assembly of thylakoid membrane protein complexes and plays a critical role for chloroplast development in Arabidopsis. However, the biological function of ALB3 homologs in rice, OsALB3, remains elusive. Here, we identified a rice mutant, yellow leaf and lethal1 (yll1), that displayed yellow leaves and died at the seedling stage. The content of chlorophyll in yll1, compared with wild type, was significantly decreased. Transmission electron microscopy observation shows that the chloroplast of yll1 lacks thylakoid membranes. The causal mutation, which is located in OsALB3, was isolated by Mutmap+ combined with a simple mutation filtering process. Knockout of OsALB3 leads to yellow leaves and seedling lethality, mimicking the phenotype of yll1. OsALB3 is widely expressed and OsALB3 is chloroplast-localized. Moreover, the content of light-harvesting chlorophyll-binding proteins in yll1 is reduced. Together, our study demonstrated the essential role of OsALB3 in chloroplast development and provided clues to the possible conserved molecular function of ALB3 in rice.

TI17, a novel compound, exerts anti-MM activity by impairing Trip13 function of DSBs repair and enhancing DNA damage.

Thyroid hormone receptor interacting protein 13 (Trip13) is an AAA-ATPase that regulates the assembly or disassembly protein complexes and mediates Double-strand breaks (DSBs) repair. Overexpression of Trip13 has been detected in many cancers and is associated with myeloma progression, disease relapse and poor prognosis inmultiple myeloma (MM). We have identified a small molecular, TI17, through a parallel compound-centric approach, which specifically targets Trip13. To identify whether TI17 targeted Trip13, pull-down and nuclear magnetic resonance spectroscopy (NMR) assays were performed. Cell counting kit-8, clone formation, apoptosis and cell cycle assays were applied to investigate the effects of TI17. We also utilized a mouse model to investigate the effects of TI17 in vivo. TI17 effectively inhibited the proliferation of MM cells, and induced the cycle arrest and apoptosis of MM cells. Furthermore, treatment with TI17 abrogates tumor growth and has no apparent side effects in mouse xenograft models. TI17 specifically impaired Trip13 function of DSBs repair and enhanced DNA damage responses in MM. Combining with melphalan or HDAC inhibitor panobinostat triggers synergistic anti-MM effect. Our study suggests that TI17 could be acted as a specific inhibitor of Trip13 and supports a preclinical proof of concept for therapeutic targeting of Trip13 in MM.