Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase.

How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.

Endocytosis-Like Vesicle Fission Mediated by a Membrane-Expanding Molecular Machine Enables Virus Encapsulation for In Vivo Delivery.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Achilles Tendon Thickening as a Risk Factor of Cardiovascular Events after Percutaneous Coronary Intervention.

AIM: The Achilles tendon (AT) thickening may be affected by several factors (e.g., lipid disorders or age). This study aims to determine the prevalence rate of AT thickening in patients with coronary artery disease (CAD) and investigate the correlation between AT thickening and the incidence of major adverse cardiovascular events (MACE) after percutaneous coronary intervention (PCI). METHODS: The clinical records of 887 patients who had undergone successful PCI and measured Achilles tendon thickness (ATT) using soft X-ray radiographs were retrospectively examined. Subjects were divided into two groups depending on the presence or absence of AT thickening. AT thickening was defined as having ATT of ＞8.0 and ＞7.5 mm in men and women, respectively. Among the two groups, the incidence of MACE was measured for a maximum of 5 years after PCI. MACE was defined as cardiovascular mortality, nonfatal myocardial infarction, or revascularization due to restenosis or the increase of stenosis in other lesions. RESULTS: This study found that 241 (27.2%) patients have AT thickening. Patients with AT thickening had higher low-density lipoprotein cholesterol (LDL-C) levels. In addition, the Kaplan-Meier curve with a log-rank test demonstrated that patients with AT thickening had a significantly higher incidence of MACE. Furthermore, the multivariate analysis indicated that the presence of AT thickening was independently correlated with the incidence of MACE after PCI. CONCLUSION: A high percentage of patients with CAD were found to have AT thickening. In addition, the presence of AT thickening was significantly associated with a higher incidence of MACE, independent of LDL-C levels.

Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function.

Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.

Stabilization of bicelles using metal-binding peptide for extended blood circulation.

A metal-binding peptide appending cholic acid, Chol-MBP, formed bicelles by mixing with 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC). Coordination of Chol-MBP with Cu2+ stabilized DPPC bicelles against dilution and contamination of serum proteins, enabling extended blood circulation. This study demonstrates an effective supramolecular design of phospholipid bicelles with enhanced stability useful for membrane-based biomaterials.

Simultaneous evaluation of fatty acid and triglycerides after percutaneous coronary intervention.

BACKGROUND: Clinical studies have shown that the ratio of eicosapentaenoic acid to arachidonic acid (EPA/AA ratio) as well as the triglyceride (TG) levels can be considered as independent risk factors for cardiovascular diseases. The aim of this study was to investigate whether simultaneous evaluation of the EPA/AA ratio and TG level can affect the incidence of cardiovascular events after percutaneous coronary intervention (PCI). METHODS AND RESULTS: We retrospectively examined the clinical records of 1585 patients who underwent successful PCI for acute coronary syndrome or stable angina. They were divided into four categories based on an EPA/AA ratio of 0.4 and a TG level of 150 mg/dl (a method termed the "Fatty Acid Window"). Among the four categories, the incidence of major adverse cardiac events (MACE) was measured for a maximum of five years after PCI. MACE was defined as cardiac death, non-fatal myocardial infarction, or revascularization due to new coronary stenosis or restenosis. The Kaplan-Meier method and the Cox proportional hazards regression analysis demonstrated that patients with both lower EPA/AA ratios and higher TG levels had a significantly higher incidence of MACE. In addition, patients with either lower EPA/AA ratios or higher TG levels also had a higher incidence of MACE compared to patients with both higher EPA/AA ratios and lower TG levels. CONCLUSION: Evaluating both EPA/AA ratios and TG levels, a method termed the "Fatty Acid Window", can be useful in predicting the occurrence of cardiovascular events after PCI.

Heartbeat tracking task performance, an indicator of interoceptive accuracy, is associated with improvement of exercise tolerance in patients undergoing home-based cardiac rehabilitation.

Aims: Interoception is the sensing function of physiological conditions and is crucial in self-regulation and decision-making. We examined the association of heartbeat tracking task performance, an indicator of interoceptive accuracy, with the degree of improvement in exercise tolerance in patients undergoing home-based cardiac rehabilitation. Methods and results: Participants underwent baseline peak oxygen uptake (VO2) measurements and a heartbeat tracking task. The heartbeat tracking task score varies between 0 and 1, with higher scores indicating a better heartbeat perception. After 6 months of home-based exercise training, peak VO2 was measured again, and the percentage change (%Δ peak VO2) relative to the peak VO2 at baseline was calculated. Univariate regression analysis was performed to examine the association between %Δ peak VO2 and the heartbeat tracking task score. Multiple regression analysis was performed to determine the predictors of %Δ peak VO2. Of 120 participants, 100 patients (age 65.9 ± 11.9 years; 86% male) were included. There was a significant positive association between %Δ peak VO2 and the heartbeat tracking task score at baseline (R 2 = 0.236, P < 0.001). In multiple regression analysis, the percentage of measured peak VO2 to the predicted value (%predicted peak VO2) (ß = -0.248, P = 0.002), exercise adherence (ß = 0.364, P < 0.001), and heartbeat tracking task score at baseline (ß = 0.372, P < 0.001) were significantly associated with %Δ peak VO2. Conclusions: Heartbeat tracking task performance, an indicator of interoceptive accuracy, at baseline is associated with the degree of improvement in exercise tolerance.

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2.

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Relationship between serum uric acid levels and the incidence of cardiovascular events after percutaneous coronary intervention.

BACKGROUND: The role of serum uric acid (SUA) as an independent risk factor for coronary artery disease remains unclear. The aim of this study was to investigate whether the SUA levels could affect the incidence of major adverse cardiac events (MACE) after percutaneous coronary intervention (PCI). METHODS AND RESULTS: We retrospectively examined the clinical records of 1,949 patients who underwent successful PCI. First, they were divided into two groups based on an SUA level of 7.0mg/dl. Among the two groups, the incidence of MACE was measured for a maximum of 5 years after PCI. Next, we divided them into 6 groups at SUA intervals of 1.0mg/dl and estimated the hazard ratios of each group. The Kaplan-Meier curve demonstrated that patients with SUA levels of >7.0mg/dl had a higher incidence of MACE than those with 7.0mg/dl or less. However, according to the multivariate analysis, the SUA level was not significantly correlated with the incidence of MACE because other factors could strongly affect it. Meanwhile, the group with SUA levels between 4.1-5.0mg/dl had a lower hazard ratio compared to groups with SUA levels of more than 5.1mg/dl. However, the hazard ratio of the group with SUA levels of 4.0mg or less was not lower than that of the group with SUA levels of 4.1-5.0mg/dl. Even after adjustment for several parameters, nearly the same results before adjustment were obtained for the hazard ratios of each group. CONCLUSION: The present study demonstrated that the SUA level was one of the most valuable predictors of cardiovascular events after PCI, with elevated SUA levels adversely affecting secondary prevention.

Mid-arm muscle circumference as an indicator of exercise tolerance in chronic heart failure.

AIM: Skeletal muscle mass is associated with exercise tolerance in patients with chronic heart failure (CHF). Anthropometric indicators are used to evaluate skeletal muscle mass, as these can be easily assessed in clinical practice. However, the association between anthropometric indicators and exercise tolerance is unclear. This study aimed to investigate the association between anthropometric indicators and exercise tolerance in CHF patients. METHODS: We evaluated 69 patients with CHF. Mid-arm circumference, mid-arm muscle circumference (MAMC), calf circumference and body mass index were measured as the anthropometric indicators. Exercise tolerance was evaluated according to the peak oxygen uptake (VO2 ). Correlation analyses were carried out to determine the association between peak VO2 and anthropometric indicators. Furthermore, univariate and multiple regression analyses were carried out using peak VO2 as the dependent variable, and age, male, left ventricular ejection fraction, angiotensin II receptor blocker or angiotensin converting enzyme inhibitor, diuretics, B-type natriuretic peptide, estimated glomerular filtration rate, hemoglobin and anthropometric indicators as the independent variables. RESULTS: There were significant positive correlations between the peak VO2 and mid-arm circumference (rs = 0.378, P = 0.001), MAMC (r = 0.634, P < 0.001) and calf circumference (r = 0.292, P = 0.015). In multiple regression analysis, MAMC (ß = 0.721, P < 0.001) and estimated glomerular filtration rate (ß = 0.279, P = 0.007) were independent factors associated with peak VO2 . CONCLUSIONS: MAMC is independently associated with peak VO2 in CHF patients. Thus, MAMC could be an indicator of exercise tolerance, which is closely related to the severity and prognosis of CHF. Geriatr Gerontol Int 2021; 21: 411-415.

Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV.

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair.

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)- and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

Erratum: Author Correction: High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

[This corrects the article DOI: 10.1038/s42003-019-0587-z.].

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

Parkinson's disease-associated mutations in the GTPase domain of LRRK2 impair its nucleotide-dependent conformational dynamics.

Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.

A practical method for efficient and optimal production of Seleno-methionine-labeled recombinant protein complexes in the insect cells.

The use of Seleno-methionine (SeMet) incorporated protein crystals for single or multi-wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared with native protein expression.

A protein-protein interaction underlies the molecular basis for substrate recognition by an adenosine-to-inosine RNA-editing enzyme.

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors.

Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

Impact of nonfasting triglycerides/high-density lipoprotein cholesterol ratio on secondary prevention in patients treated with statins.

BACKGROUND: Some studies have demonstrated that low-density lipoprotein cholesterol (LDL-C) lowering therapy is one of the most important strategies to prevent coronary artery disease. Also, serum triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) are recognized as independent risk factors of cardiovascular diseases. The aim of this study was to investigate whether the nonfasting TG/HDL-C ratio could affect the incidence of cardiovascular events after percutaneous coronary intervention (PCI) even in patients treated with statins. METHODS AND RESULTS: One thousand one hundred seventy consecutive patients were enrolled, all of whom underwent successful PCI for acute coronary syndrome or stable angina and continued statin treatments after PCI. They were equally divided into three groups on the basis of a nonfasting TG/HDL-C ratio 3 months after PCI. Among these groups, the incidence of major adverse cardiac events (MACE) was measured during a maximum of 5 years after PCI. MACE was defined as cardiac death, nonfatal myocardial infarction, revascularization due to new stenosis or restenosis. Kaplan-Meier analysis demonstrated that patients with higher TG/HDL-C ratio had a significantly higher incidence of MACE than other groups (p<0.001). In addition, Cox proportional hazards regression analysis indicated that the nonfasting TG/HDL-C ratio was significantly correlated with the incidence of MACE. CONCLUSION: The nonfasting TG/HDL-C ratio was a valuable predictor of cardiovascular events after PCI in patients treated with statins.