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Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a proapoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2 dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c + cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment of the AKT/mTORC1 pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design., (© 2024 The Protein Society.)

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1β (IL-1β). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1β. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1β. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1β by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1β, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many proteases recognize their substrates with high specificities, with this in mind, it should theoretically be possible to utilize the substrate binding cleft of a protease as a scaffold to engineer an affinity reagent. In this study, we sought to develop reagents that would differentiate between substrates and products of proteolysis, based on a caspase 7 scaffold. Firstly, we engineered a form of caspase 7 that can undergo conversion to a substrate binding conformation without catalysis. Seeking to generate a product-only trap, we further engineered this construct by incorporating mutations that compensate for the generation of a negative charge in the neo C terminus of a newly generated product. This was accomplished with only three substitutions within the substrate binding cleft. Moreover, the affinity of the product trap for peptides was comparable to the affinity of caspase 7 to parental substrates. Finally, generation of a hybrid fluorescent protein with the product trap provided a reagent that specifically recognized apoptotic cells and highlights the versatility of such an approach in developing affinity and imaging agents for a variety of cysteine and serine proteases., (© 2020 Federation of European Biochemical Societies.)

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Bibo-Verdugo et al.)

Proteases play critical roles in virtually all biological processes, including proliferation, cell death and survival, protein turnover, and migration. However, when dysregulated, these enzymes contribute to the progression of multiple diseases, with cancer, neurodegenerative disorders, inflammation, and blood disorders being the most prominent examples. For a long time, disease-associated proteases have been used for the activation of various prodrugs due to their well-characterized catalytic activity and ability to selectively cleave only those substrates that strictly correspond with their active site architecture. To date, versatile peptide sequences that are cleaved by proteases in a site-specific manner have been utilized as bioactive linkers for the targeted delivery of multiple types of cargo, including fluorescent dyes, photosensitizers, cytotoxic drugs, antibiotics, and pro-antibodies. This platform is highly adaptive, as multiple protease-labile conjugates have already been developed, some of which are currently in clinical use for cancer treatment. In this review, recent advancements in the development of novel protease-cleavable linkers for selective drug delivery are described. Moreover, the current limitations regarding the selectivity of linkers are discussed, and the future perspectives that rely on the application of unnatural amino acids for the development of highly selective peptide linkers are also presented., (© 2020 Federation of European Biochemical Societies.)

Background and Aims: We have previously found increased levels of the cysteine protease legumain in plasma and plaques from patients with carotid atherosclerosis. This study further investigated legumain during acute cardiovascular events., Methods: Circulating levels of legumain from patients and legumain released from platelets were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting were used to study expression, while localization was visualized by immunohistochemistry., Results: In the SUMMIT Malmö cohort (n = 339 with or without type 2 diabetes and/or cardiovascular disease [CVD], and 64 healthy controls), the levels of circulating legumain were associated with the presence of CVD in non-diabetics, with no relation to outcome. In symptomatic carotid plaques and in samples from both coronary and intracerebral thrombi obtained during acute cardiovascular events, legumain was co-localized with macrophages in the same regions as platelets. In vitro, legumain was shown to be present in and released from platelets upon activation. In addition, THP-1 macrophages exposed to releasate from activated platelets showed increased legumain expression. Interestingly, primary peripheral blood mononuclear cells stimulated with recombinant legumain promoted anti-inflammatory responses. Finally, in a STEMI population (POSTEMI; n = 272), patients had significantly higher circulating legumain before and immediately after percutaneous coronary intervention compared with healthy controls (n = 67), and high levels were associated with improved outcome., Conclusions: Our data demonstrate for the first time that legumain is upregulated during acute cardiovascular events and is associated with improved outcome., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This screen uncovered previously unknown caspase-2 peptidyl substrate preferences, which were further used to develop caspase-2 selective fluorogenic substrates and covalent, irreversible AOMK inhibitors. Finally, we used the champion inhibitor (NH-23-C2) in reversine-treated HCT-116 colon cancer cells to selectively block caspase-2 activity and caspase-2-mediated MDM-2 cleavage. In addition, we showed that NH-23-C2 does not block caspase-3 or caspase-8, which makes it a powerful chemical tool to dissect the true role of caspase-2 in various biological setups.

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes, it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European BiochemicalSocieties.)

The zymogen form of circulating Factor VII activating protease (FSAP) is activated by histones that are released as a consequence of tissue damage or excessive inflammation. This is likely to have consequences in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To investigate the existence, as well as the concentration of active FSAP (FSAPa) in complex biological systems an active site probe is needed. We used Hybrid Combinatorial Substrate Library (HyCoSuL) to screen for natural and unnatural amino acids that specifically bind to P4-P2 pockets of FSAPa. This information was used to designing a fluorogenic substrate (Ac-Pro-DTyr-Lys-Arg-ACC) as well as an irreversible, fluorogenic activity-based probe Cy5-6-Ahx-Pro-DTyr-Lys-Arg P (OPh) 2 . In normal human plasma the probe showed very low non-specific reactivity with some plasma proteins but upon activation of pro-FSAP with histones, strong labelling of FSAPa was observed. This labelling could be inhibited by aprotinin and was not found in the plasma of a subject that was homozygous for a polymorphism, which leads to loss of activity, or in plasma that was depleted of FSAP by antibodies. This 2nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1st generation substrate and a much higher selectivity for FSAPa over other plasma proteases. This substrate and probe can be useful to detect and localize FSAPa in normal and pathological tissue and plasma to gain more insight into its functions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Human cysteine cathepsins constitute an 11-membered family of proteases responsible for degradation of proteins in cellular endosomal-lysosomal compartments as such, they play important roles in antigen processing, cellular stress signaling, autophagy, and senescence. Moreover, for many years these enzymes were also linked to tumor growth, invasion, angiogenesis and metastasis when upregulated. Individual biological roles of each cathepsin are difficult to establish, because of their redundancy and similar substrate specificities. Selective chemical tools that enable imaging of individual cathepsin activities in living cells, tumors, and the tumor microenvironment may provide a better insight into their functions. In this work, we used HyCoSuL technology to profile the substrate specificity of human cathepsin B. The use of unnatural amino acids in the substrate library enabled us to uncover the broad cathepsin B preferences that we utilized to design highly-selective substrates and fluorescent activity-based probes (ABPs). We further demonstrated that Cy5-labeled MP-CB-2 probe can selectively label cathepsin B in eighteen cancer cell lines tested, making this ABP highly suitable for other biological setups. Moreover, using Cy5-labelled MP-CB-2 we were able to demonstrate by fluorescence microscopy that in cancer cells cathepsins B and L share overlapping, but not identical subcellular localization., (This journal is © The Royal Society of Chemistry 2019.)

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

The ability to label active caspase-3 represents a useful pharmacodynamic strategy to determine the efficacy of anti-tumour drugs. Activity-based probes (ABPs) provide a method for the labelling of activated caspases and the recent development of hybrid combinatorial substrate libraries (HyCoSuL) has allowed for the generation of highly selective ABPs to discriminately label these proteases. Here using this approach, a novel caspase-3 selective ABP (CS1) has been developed and validated in apoptotic cells to selectively bind caspase-3 over the closely related caspase-7. However, a critical bottleneck for ABPs is their cell penetrance and therefore this cell-impermeable CS1 probe was subsequently formulated into PLGA-based nanoparticles (CS1-NPs). We demonstrate the ability of these particles to be taken up by the cells and facilitate intracellular delivery of the ABP to effectively label caspase 3 in response to apoptotic stimuli. This work forms the foundation of a novel approach for the labelling of caspase 3 and may have downstream utility to measure real time apoptosis in tumours and other organs.

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: There are inconsistent data about the role of serum phospholipid fatty acid composition in patients with type 2 diabetes (T2DM) and atherosclerotic cardiovascular disease (ASCVD). The aim of the study was to investigate the relationship between serum phospholipid fatty acid composition, systemic low-grade inflammation, and glycemic control in high-risk T2DM patients., Methods: Seventy-four patients (26% women, mean age 65.6 ± 6.8 years) with T2DM (median diabetes duration 10 years) and documented ASCVD (74 with coronary artery disease, 26 with peripheral arterial disease) were enrolled in the study. Baseline HbA 1c was estimated using turbidimetric inhibition immunoassay. According to the median value of HbA 1c the patients were grouped into those with HbA 1c  < 7.0% (< 53 mmol/mol) (n = 38) and those with HbA 1c  ≥ 7.0% (≥ 53 mmol/mol) (n = 36). Serum phospholipid fatty acids were measured with gas chromatography., Results: Patients with HbA 1c  ≥ 7.0%, compared with those with HbA 1c  < 7.0% had similar composition of saturated and monounsaturated fatty acids in serum phospholipids, but had higher concentrations of linoleic acid (LA) and higher n-6/n-3 polyunsaturated fatty acid (PUFA) ratio as well as lower levels of eicosapentaenoic acid (EPA), total n-3 PUFAs, and the EPA/arachidonic acid ratio. We found that LA (r = 0.25; p = 0.03) and n-6/n-3 PUFA ratio (r = 0.28; p = 0.02) were positively correlated with HbA 1c . Multivariate logistic regression analysis showed that n-6/n-3 PUFA ratio, hsCRP and T2DM duration were independent predictors of worse glycemic control in patients with T2DM and ASCVD., Conclusions: This study showed that glycemic control in high-risk T2DM patients with ASCVD was significantly associated with unfavorable serum phospholipid n-6/n-3 PUFA ratio and greater systemic inflammation.