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Secretory proteins are critically dependent on the correct processing of their signal sequence by the signal peptidase complex (SPC). This step, which is essential for the proper folding and localization of proteins in eukaryotic cells, is still not fully understood. In eukaryotes, the SPC comprises four evolutionarily conserved membrane subunits (Spc1-3 and Sec11). Here, we investigated the role of Spc2, examining SPC cleavage efficiency on various models and natural signal sequences in yeast cells depleted of or with mutations in Spc2. Our data show that discrimination between substrates and identification of the cleavage site by SPC is compromised when Spc2 is absent or mutated. Molecular dynamics simulation of the yeast SPC AlphaFold2-Multimer model indicates that membrane thinning at the center of SPC is reduced without Spc2, suggesting a molecular explanation for the altered substrate recognition properties of SPC lacking Spc2. These results provide new insights into the molecular mechanisms by which SPC governs protein biogenesis., (© 2024 Chung et al.)

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Type 2 diabetes (T2D) is a rising pandemic worldwide. Diet and lifestyle changes are typically the first intervention for T2D. When this intervention fails, the biguanide metformin is the most common pharmaceutical therapy. Yet its full mechanisms of action remain unknown. In this work, we applied an ultrahigh resolution, mass spectrometry-based platform for untargeted plasma metabolomics to human plasma samples from a case-control observational study of nondiabetic and well-controlled T2D subjects, the latter treated conservatively with metformin or diet and lifestyle changes only. No statistically significant differences existed in baseline demographic parameters, glucose control, or clinical markers of cardiovascular disease risk between the two T2D groups, which we hypothesized would allow the identification of circulating metabolites independently associated with treatment modality. Over 3000 blank-reduced metabolic features were detected, with the majority of annotated features being lipids or lipid-like molecules. Altered abundance of multiple fatty acids and phospholipids were found in T2D subjects treated with diet and lifestyle changes as compared with nondiabetic subjects, changes that were often reversed by metformin. Our findings provide direct evidence that metformin monotherapy alters the human plasma lipidome independent of T2D disease control and support a potential cardioprotective effect of metformin worthy of future study. SIGNIFICANCE STATEMENT: This work provides important new information on the systemic effects of metformin in type 2 diabetic subjects. We observed significant changes in the plasma lipidome with metformin therapy, with metabolite classes previously associated with cardiovascular disease risk significantly reduced as compared to diet and lifestyle changes. While cardiovascular disease risk was not a primary outcome of our study, our results provide a jumping-off point for future work into the cardioprotective effects of metformin, even in well-controlled type 2 diabetes., (U.S. Government work not protected by U.S. copyright.)

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal ⍺-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates through tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome-derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Over half of patients with type 2 diabetes (T2D) are unable to achieve blood glucose targets despite therapeutic compliance, significantly increasing their risk of long-term complications. Discovering ways to identify and properly treat these individuals is a critical problem in the field. The arachidonic acid metabolite, prostaglandin E 2 (PGE 2 ), has shown great promise as a biomarker of β-cell dysfunction in T2D. PGE 2 synthesis, secretion, and downstream signaling are all upregulated in pancreatic islets isolated from T2D mice and human organ donors. In these islets, preventing β-cell PGE 2 signaling via a prostaglandin EP3 receptor antagonist significantly improves their glucose-stimulated and hormone-potentiated insulin secretion response. In this clinical cohort study, 167 participants, 35 non-diabetic, and 132 with T2D, were recruited from the University of Wisconsin Hospital and Clinics. At enrollment, a standard set of demographic, biometric, and clinical measurements were performed to quantify obesity status and glucose control. C reactive protein was measured to exclude acute inflammation/illness, and white cell count (WBC), erythrocyte sedimentation rate (ESR), and fasting triglycerides were used as markers of systemic inflammation. Finally, a plasma sample for research was used to determine circulating PGE 2 metabolite (PGEM) levels. At baseline, PGEM levels were not correlated with WBC and triglycerides, only weakly correlated with ESR, and were the strongest predictor of T2D disease status. One year after enrollment, blood glucose management was assessed by chart review, with a clinically-relevant change in hemoglobin A1c (HbA1c) defined as ≥0.5%. PGEM levels were strongly predictive of therapeutic response, independent of age, obesity, glucose control, and systemic inflammation at enrollment. Our results provide strong support for future research in this area.

Proteolytic cleavage of cell surface proteins triggers critical processes including cell-cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2 , induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies., Competing Interests: The authors declare the following competing financial interest(s): J.A.W. and K.S. filed a provisional patent on the cell surface N-terminomics technology., (© 2022 The Authors. Published by American Chemical Society.)

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8 + surface proteome (surfaceome). Surprisingly, coculturing primary CD8 + T cells with Tregs only modestly affected the CD8 + surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8 + surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4 + T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function., Competing Interests: Conflict of interest A. A. is a cofounder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation; a consultant for SPARC, Bluestar, ProLynx, Earli, Cura, GenVivo and GSK; a member of the Scientific Advisory Board of Genentech, GLAdiator, Circle and Cambridge Science Corporation; receives grant/research support from SPARC and AstraZeneca; holds patents on the use of PARP inhibitors held jointly with AstraZeneca, which he has benefitted financially (and may do so in the future). A. M. is cofounder, member of the Boards of Directors and member of Scientific Advisory Boards of Spotlight Therapeutics and Arsenal Biosciences. A. M. has served as an advisor to Juno Therapeutics, was a member of the Scientific Advisory Board at PACT Pharma and was an advisor to Trizell. A. M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. A. M. owns stock in Arsenal Biosciences, Spotlight Therapeutics and PACT Pharma. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem. J. A. W. is cofounder of Soteria Therapeutics, is on the Scientific Advisory Board of Jnana Therapeutics, Inception Therapeutics, IgGenix Inc, Red Tree Capital, Spotlight Therapeutics, Inzen Therapeutics, and receives research support from Bristol-Myers-Squibb, TRex Bio and Merck, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Apoptosis is a cell death program that is executed by the caspases, a family of cysteine proteases that typically cleave after aspartate residues during a proteolytic cascade that systematically dismantles the dying cell. Extensive signaling crosstalk occurs between caspase-mediated proteolysis and kinase-mediated phosphorylation, enabling integration of signals from multiple pathways into the decision to commit to apoptosis. A new study from Maluch et al. examines how phosphorylation within caspase cleavage sites impacts the efficiency of substrate cleavage. The results demonstrate that while phosphorylation in close proximity to the scissile bond is generally inhibitory, it does not necessarily abrogate substrate cleavage, but instead attenuates the rate. In some cases, this inhibition can be overcome by additional favorable substrate features. These findings suggest potential nuanced physiological roles for phosphorylation of caspase substrates with exciting implications for targeting caspases with chemical probes and therapeutics., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Elevated islet production of prostaglandin E 2 (PGE 2 ), an arachidonic acid metabolite, and expression of prostaglandin E 2 receptor subtype EP3 (EP3) are well-known contributors to the β-cell dysfunction of type 2 diabetes (T2D). Yet, many of the same pathophysiological conditions exist in obesity, and little is known about how the PGE 2 production and signaling pathway influences nondiabetic β-cell function. In this work, plasma arachidonic acid and PGE 2 metabolite levels were quantified in a cohort of nondiabetic and T2D human subjects to identify their relationship with glycemic control, obesity, and systemic inflammation. In order to link these findings to processes happening at the islet level, cadaveric human islets were subject to gene expression and functional assays. Interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA levels, but not those of EP3, positively correlated with donor body mass index (BMI). IL-6 expression also strongly correlated with the expression of COX-2 and other PGE 2 synthetic pathway genes. Insulin secretion assays using an EP3-specific antagonist confirmed functionally relevant upregulation of PGE 2 production. Yet, islets from obese donors were not dysfunctional, secreting just as much insulin in basal and stimulatory conditions as those from nonobese donors as a percent of content. Islet insulin content, on the other hand, was increased with both donor BMI and islet COX-2 expression, while EP3 expression was unaffected. We conclude that upregulated islet PGE 2 production may be part of the β-cell adaption response to obesity and insulin resistance that only becomes dysfunctional when both ligand and receptor are highly expressed in T2D., Competing Interests: The authors declare the following competing financial interest(s): R.J.F., H.K.S., C.P., B.W., S.P., A.R., J.M.H., A.L.B., D.C.P., R.N., M.D., M.P., C.E.K., E.D.C., Y.G., D.B.D., and M.E.K. declare that they have no conflicts of interest with the contents of this article. NAT is currently a Nanomedicine Innovation Center LLC employee (46701 Commerce Center Drive, Plymouth, MI 48170). AMW is currently an Allena Pharmaceuticals employee (One Newton Executive Park, Suite 202, Newton, MA 02462). This work was completed in full during their research training with Dr. Kimple and is not related to their current positions., (© 2021 American Chemical Society.)

Mass spectrometry-based proteomics has been used successfully to identify substrates for proteases. Identification of protease substrates at the cell surface, however, can be challenging since cleavages are less abundant compared to other cellular events. Precise methods are required to delineate cleavage events that take place in these compartmentalized areas. This article by up-and-coming scientist Dr. Amy Weeks, an Assistant Professor at the University of Wisconsin-Madison, provides an overview of methods developed to identify protease substrates and their cleavage sites at the membrane. An overview is presented with the pros and cons for each method and in particular the N-terminomics subtiligase-TM method, developed by Dr. Weeks as a postdoctoral fellow in the lab of Dr. Jim Wells at University of California, San Francisco, is described in detail. Subtiligase-TM is a genetically engineered subtilisin protease variant that acts to biotinylate newly generated N termini, hence revealing new cleavage events in the presence of a specific enzyme, and furthermore can precisely identify the cleavage P1 site. Importantly, this proteomics method is compatible with living cells. This method will open the door to understanding protein shedding events at the biological membrane controlled by proteases to regulate biological processes.

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease., Competing Interests: Competing interest statement: A.M.W. and J.A.W. and the Regents of the University of California have filed a patent application (US Provisional Patent Application 62/398,898) related to engineered subtiligase variants.

Post-translational modifications, complex formation, subcellular localization, and cell-type-specific expression create functionally distinct protein subpopulations that enable living systems to execute rapid and precise responses to changing conditions. Systems-level analysis of these subproteomes remains challenging, requiring preservation of spatial information or enrichment of species that are transient and present at low abundance. Engineered proteins have emerged as important tools for selective proteomics based on their capacity for highly specific molecular recognition and their genetic targetability. Here, we focus on new developments in protein engineering for selective proteomics of post-translational modifications, protein complexes, subcellular compartments, and cell types. We also address remaining challenges and future opportunities to integrate engineered protein tools across different subproteome scales to map the proteome with unprecedented depth and detail., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Enzymes that catalyze peptide ligation are powerful tools for site-specific protein bioconjugation and the study of cellular signaling. Peptide ligases can be divided into two classes: proteases that have been engineered to favor peptide ligation, and protease-related enzymes with naturally evolved peptide ligation activity. Here, we provide a review of key natural peptide ligases and proteases engineered to favor peptide ligation activity. We cover the protein engineering approaches used to generate and improve these tools, along with recent biological applications, advantages, and limitations associated with each enzyme. Finally, we address future challenges and opportunities for further development of peptide ligases as tools for biological research., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Subtiligase-catalyzed peptide ligation is a powerful approach for site-specific protein bioconjugation, synthesis and semisynthesis of proteins and peptides, and chemoproteomic analysis of cellular N termini. Here, we provide a comprehensive review of the subtiligase technology, including its development, applications, and impacts on protein science. We highlight key advantages and limitations of the tool and compare it to other peptide ligase enzymes. Finally, we provide a perspective on future applications and challenges and how they may be addressed.

Subtiligase is a powerful enzymatic tool for N-terminal modification of proteins and peptides. In a typical subtiligase-catalyzed N-terminal modification reaction, a peptide ester donor substrate is ligated onto the unblocked N terminus of a protein, resulting in the exchange of the ester bond in the donor substrate for an amide bond between the donor substrate and protein N terminus. Using this strategy, new chemical probes and payloads, such as fluorophores, affinity handles, cytotoxic drugs, and reactive functional groups, can be introduced site-specifically into proteins. While the efficiency of this reaction depends on the sequences to be ligated, a panel of mutants was recently developed that expands the scope of substrate sequences that are suitable for subtiligase modification. This article outlines the steps for applying subtiligase or specificity variants for both site-specific bioconjugation of purified proteins and for global modification of cellular N termini to enable their sequencing by tandem mass spectrometry. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Subtiligase-catalyzed site-specific protein bioconjugation Support Protocol 1: Expression and purification of subtiligase-His 6 Support Protocol 2: Subtiligase substrate synthesis Basic Protocol 2: Subtiligase N terminomics using a cocktail of subtiligase specificity mutants., (© 2020 John Wiley & Sons, Inc.)

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from Streptomyces cattleya thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 10 6 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding ( K D ). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity., Competing Interests: The authors declare no conflict of interest.

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Wild-type AMPA receptors display a characteristic rapidly desensitizing phenotype. Many studies point to the dimer interface between pairs of extracellular ligand binding domains as the key region controlling the rate at which the receptors desensitize. However, mutations at the extracellular end of the pore-forming regions (near the putative ion channel gate) have also been shown to alter desensitization. Here we report the behavior of single GluA4 receptors carrying one of two mutations that greatly reduce desensitization at the level of ensemble currents: the dimer interface mutation L484Y and the Lurcher mutation (A623T, GluA4-Lc) in the extracellular end of M3 (the second true transmembrane helix). Analysis of unitary currents in patches with just one active receptor showed that each mutation greatly prolongs bursts of openings without prolonging the apparent duration of individual openings. Each mutation decreases the frequency with which individual receptors visit desensitized states, but both mutant receptors still desensitize multiple times per second. Cyclothiazide (CTZ) reduced desensitization of wild-type receptors and both types of mutant receptor. Analysis of shut-time distributions revealed a form of short-lived desensitization that was resistant to CTZ and was especially prominent for GluA4-Lc receptors. Despite reducing desensitization of GluA4 L484Y receptors, CTZ decreased the amplitude of ensemble currents through GluA2 and GluA4 LY receptor mutants. Single-channel analysis and comparison of the GluA2 L483Y ligand binding domain dimer in complex with glutamate with and without CTZ is consistent with the conclusion that CTZ binding to the dimer interface prevents effects of the LY mutation to modulate receptor activation, resulting in a reduction in the prevalence of large-conductance substates that accounts for the decrease in ensemble current amplitudes. Together, the results show that similar nondesensitizing AMPA-receptor phenotypes of population currents can arise from distinct underlying molecular mechanisms that produce different types of unitary activity., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes., (Copyright © 2017, American Association for the Advancement of Science.)

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five "subsites" which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites "A" and "B" of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH-rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[(2)H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-(2)H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts.

The investigation of unique chemical phenotypes has led to the discovery of enzymes with interesting behaviors that allow us to explore unusual function. The organofluorine-producing microbe Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that demonstrates a surprisingly high level of discrimination for a single fluorine substituent on its substrate compared with the cellularly abundant hydrogen analog, acetyl-CoA. In this report, we show that the high selectivity of FlK is achieved through catalysis rather than molecular recognition, where deprotonation at the C(α) position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10(4)-fold compared with the nonfluorinated congener. These studies provide insight into mechanisms of catalytic selectivity in a native system where the existence of two reaction pathways determines substrate rather than product selection.

Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya's resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host's metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products.

The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these steps are quite diverse in their structural and biochemical features and are important for controlling differences at the cellular level. One of the key steps in the fatty acid elongation cycle is the enoyl-ACP (CoA) reductase function that drives the equilibrium forward toward chain extension. In this work, we report the structural and biochemical characterization of the trans-enoyl-CoA reductase from Treponema denticola (tdTer), which has been utilized for the engineering of synthetic biofuel pathways with an order of magnitude increase in product titers compared to those of pathways constructed with other enoyl-CoA reductase components. The crystal structure of tdTer was determined to 2.00 Å resolution and shows that the Ter enzymes are distinct from members of the FabI, FabK, and FabL families but are highly similar to members of the FabV family. Further biochemical studies show that tdTer uses an ordered bi-bi mechanism initiated by binding of the NADH redox cofactor, which is consistent with the behavior of other enoyl-ACP (CoA) reductases. Mutagenesis of the substrate binding loop, characterization of enzyme activity with respect to crotonyl-CoA, hexenoyl-CoA, and dodecenoyl-CoA substrates, and product inhibition by lauroyl-CoA suggest that this region is important for controlling chain length specificity, with the major portal playing a more important role for longer chain length substrates.

At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1-3',2')1,3-oxazino(6',5'-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously.

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.

Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.

Glutamate is the predominant excitatory neurotransmitter in the central nervous system. The receptors that bind glutamate, including N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subtypes, are strongly implicated in higher cognitive processes, especially learning and memory. Loss of glutamate receptor function impairs the ability to acquire and retain information in some patients subsequent to stroke or brain injury, and positive allosteric modulators of glutamate receptors can restore learning and memory in some of these patients. Here we demonstrate that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts upon heterologous AMPA receptors via two distinct mechanisms. Low (nanomolar to micromolar) concentrations of KYNA facilitate AMPA receptor responses, whereas high (millimolar) concentrations of KYNA competitively antagonize glutamate receptors. Low concentrations of KYNA appear to increase current responses through allosteric modulation of desensitization of AMPA receptors. These findings suggest the possibility that low concentrations of endogenous KYNA acting at AMPA receptors may be a positive modulator of excitatory synaptic transmission.

Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the "hinge" in the ligand-binding core "clamshell" that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of ionotropic glutamate receptors consists of rapidly gating ion channels. Positive modulation of channel gating may slow gating kinetics through at least two distinct mechanisms, evidenced by the predominant slowing of either the rate of receptor desensitization or the rate of offset after agonist withdrawal (deactivation). This study compares the actions of two positive allosteric modulators [cyclothiazide, which modulates desensitization, and 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546), which modulates deactivation] in a mutant shown previously to impede modulation by cyclothiazide. These experiments test the hypothesis that the point mutation, GluR1(S493T), would also cause a loss of modulation by CX546. Wild-type GluR1 through -4 receptors were modulated by CX546, as assayed by the potentiation of steady-state currents in the Xenopus laevis oocyte expression system. CX546 potentiated steady-state currents of both splice isoforms of GluR1. Modulation by CX546 was completely abolished in GluR1(S493T) and its homolog, GluR2(S497T), although this mutation did not affect apparent agonist affinity in the absence of CX546. Thus, the GluR1(S493T) mutation has a similar impairment of modulation by either cyclothiazide or CX546, indicating that some residues at the subunit interface of glutamate receptors play an important role in channel deactivation and desensitization.

Quality of recovery after an operation is an important dimension of the patient's experience and may be related to the quality of anaesthesia care. Satisfaction with anaesthesia is a vital component of quality care but difficult to measure. We examined our database of 5672 adult patients to determine if quality of recovery is associated with satisfaction with anaesthesia and to identify the perioperative factors that might influence both these outcome measures. We found that a nine-item quality of recovery score ("QoR Score") was related to satisfaction with anaesthesia (P < 0.0005): the overall level of satisfaction was high (97.2%; median QoR Score 16); 106 patients (2.1%; median QoR Score 14) were "somewhat dissatisfied" and 32 patients (0.6%; median QoR Score 13) were "dissatisfied" with their anaesthesia care. Patients who experienced any of a number of perioperative complications had lower QoR Scores (P < 0.0005). We have further demonstrated the validity and clinical utility of the QoR Score, and in particular, its relationship to patient satisfaction in adult surgical patients.

Patient satisfaction after anaesthesia is an important outcome of hospital care. We analysed our anaesthetic database to identify potentially modifiable factors associated with dissatisfaction. At the time of analysis, our database contained information on 10,811 in-patients interviewed on the first day after operation. The major subjective outcome measure was patient satisfaction. We also measured other predetermined outcomes, such as nausea, vomiting, pain and complications. The overall level of satisfaction was high (96.8%); 246 (2.3%) patients were 'somewhat dissatisfied' and 97 (0.9%) were 'dissatisfied' with their anaesthetic care. After adjustment for patient and surgical factors, there was a strong relation between patient dissatisfaction and: (i) intraoperative awareness (odds ratio (OR) 54.9, 95% confidence intervals (CI) 15.7-191); (ii) moderate or severe postoperative pain (OR 3.94, 95% CI 3.16-4.91); (iii) severe nausea and vomiting (OR 4.09, 95% CI 3.18-5.25); and (iv) any other postoperative complications (OR 2.04, 95% CI 1.61-2.56). Several factors associated with dissatisfaction may be preventable or better treated.

Bilateral lung volume reduction surgery was introduced into Australia in 1995 for treatment of selected patients with emphysema. We present our experience of the anaesthetic management of our first 55 cases and describe factors associated with outcome. There were four postoperative deaths (7%). Mean (SD) total operation time was 231 (72) minutes. Median intensive care unit (ICU) stay was 26 hours. There was a significant improvement in postoperative lung function (FEV1, VC, 6-minute walk test, all P < 0.001). Eight patients (15%) required reintubation for respiratory failure; three of these patients subsequently died. With multivariate analysis, total operation time was the only significant predictor of length of ICU stay R2 = 0.25, P = 0.001), which itself was the only significant predictor of hospital stay duration (R2 = 0.36, P < 0.001).

Objective: To determine the accuracy and clinical utility of a continuous arterial blood gas (ABG) monitor during lung transplantation., Design: Prospective, observational cohort study., Setting: University hospital., Participants: Eleven patients undergoing bilateral sequential lung transplantation (BSLTx)., Interventions: Repeated ABG sampling., Measurements and Main Results: Agreement was measured by the bias (limits of agreement): pH, 0.006 (-0.10 to 0.10); PaO2, -22 mmHg (-130 to 86 mmHg); and PaCO2, -1.6 mmHg (-13.4 to 10.2 mmHg). Sensitivity and specificity of the Paratrend 7 (Biomedical Sensors, Ltd, Malvern, PA) PaO2 measurements (to detect PaO2 < 100 mmHg) were 84.6% and 97.6%, respectively., Conclusion: Continuous ABG monitoring with the Paratrend 7 shows sufficiently good agreement with laboratory blood gas analysis during BSLTx and thus is a convenient alternative to intermittent laboratory blood gas measurement. Because of the potential for significant (and sometimes rapid) acid-base disturbances, continuous ABG monitoring may have a role during most lung transplantation procedures.

The lessons learned in industry can be applied to anaesthetic departments. Progress and development are part of improving quality, and evidence is emerging that improved quality can reduce costs. Improving quality requires anaesthetic departments to develop a clear customer focus. Education, training, research and quality improvement are essential components of a quality anaesthetic department. Some of the cost reductions are achieved by development of partnerships with customers and suppliers. The emphasis is likely to shift from cost reduction to quality improvement and anaesthetic departments should anticipate this change.

Positive pressure ventilation in patients with obstructive lung disease may result in over-inflation of the relatively compliant lungs, resulting in dynamic hyperinflation (DHI). Using a crossover trial design, we compared high-frequency jet ventilation (HFJV) versus "optimal" intermittent positive pressure ventilation (IPPV) in ten patients undergoing lung transplantation for severe, end-stage obstructive lung disease. We measured haemodynamics and the degree of DHI after both modes of ventilation. There were no significant differences between IPPV and HFJV, with respect to efficiency of ventilation (PaCO2), haemodynamic effects (stroke volume, blood pressure and cardiac output), or lung hyperinflation (trapped gas volume). This study suggests that HFJV, when compared with optimal IPPV, is no better at minimizing DHI in patients with severe, end-stage obstructive lung disease.

We studied the hemodynamic effects of left atrial (LA) administration of epinephrine in 10 patients after cardiac transplantation, using a prospective, randomized, double-blind, cross-over design. After allograft implantation, a LA catheter was inserted and epinephrine infusion commenced at 100 ng.kg-1.min-1. Each trial period consisted of 20 min, with the LA and right atrial (RA) lines switched over between each period; hemodynamic measurements were taken after each time period. Whether epinephrine was administered via the RA or LA did not significantly alter hemodynamics (RA versus LA): mean (SD) arterial blood pressure 67 (7.5) vs 64 (9.5) mm Hg (P = 0.16), mean pulmonary artery pressure 22 (4.0) vs 21 (9.4) mm Hg (P = 0.67), cardiac index 3.2 (1.1) vs 3.2 (1.1) L.min-1.m-2 (P = 0.83), pulmonary vascular resistance index 308 (157) vs 345 (157) dynes.s.cm-5/m-2 (P = 0.30) or right ventricular ejection fraction 35% (11%) vs 32% (9.8%) (P = 0.23). Arterial epinephrine plasma levels were similar (P = 0.16). There was no significant pulmonary extraction of measured catecholamines. We observed no hemodynamic benefit of LA epinephrine administration. It may be that the cardiac transplantation population reacts differently compared with other cardiac surgical patients (possibly because pulmonary extraction of catecholamines is reduced). Because we did not observe a hemodynamic advantage in patients immediately after cardiac transplantation, we would not recommend the use of LA epinephrine at the dose studied.

Objectives: To review the experience of anesthesia for bilateral sequential lung transplantation (BSLTx) and describe factors associated with outcome., Design: Case series., Setting: University hospital., Participants: Sixty-four adult patients undergoing BSLTx., Interventions: Descriptive and inferential statistical analysis., Measurements and Main Results: Details of anesthetic technique, patient, and perioperative characteristics are presented. Mean (SD) lung allograft ischemic times were 320 (81) minutes for the first lung and 446 (93) minutes for the second lung. Mean (SD) duration of surgery was 8.5(2) hours, and median time to extubation was 28 hours. There was a reduction in the use of cardiopulmonary bypass, from 10 of 19 (53%) in 1992 to 1993 to 10 of 45 (22%) in 1994 to 1996, p = 0.016. There was an association between time to extubation and duration of surgery (Spearman rank correlation, p = 0.33, p = 0.008), but no association with intraoperative fluid administration (p = 0.18, p = 0.16), or inotrope requirements (p = 0.06, p = 0.65). Predictors of in-hospital mortality were preoperative renal impairment (p = 0.034), early reoperation (p = 0.005), and delay in extubation (p = 0.013); and for 12-month mortality was patient age (p = 0.01). The actuarial survival rates were 90%, 73%, and 58% at 30 days, 1 year, and 2 years, respectively., Conclusions: Anesthesia for BSLTx is a most challenging procedure, for which maintenance of tissue oxygenation and right ventricular perfusion are essential. Recent advances include use of inhaled nitric oxide, ventilator management that reduces dynamic hyperinflation, and permissive hypercapnia. Analysis of outcome from a large case series such as this enables the anesthesiologist to be more aware of the important features of anesthesia for BSLTx, as well as identify potential areas of improvement.

Recent interest in earlier tracheal extubation after coronary artery bypass graft (CABG) surgery has focused attention on the potential benefits of a propofol-based technique. We randomized 124 patients (34 with poor ventricular function) undergoing CABG surgery to receive either a propofol-based (5 mg.kg-1.h-1 prior to sternotomy, 3 mg.kg-1. h-1 thereafter; n = 58) or enflurane-based (0.2%-1.0%, n = 66) anesthetic. Induction of anesthesia consisted of fentanyl 15 micrograms/kg and midazolam 0.05 mg/kg intravenously in both groups. The enflurane group received an additional bolus of fentanyl 5 micrograms/kg prior to sternotomy and fentanyl 10 micrograms/kg with midazolam 0.1 mg/kg at commencement of cardiopulmonary bypass (CPB). Patients receiving propofol were extubated earlier (median 9.1 h versus 12.3 h, P = 0.006), although there was no difference in time to intensive care unit (ICU) discharge (both 22 h, P = 0.54). Both groups had similar hemodynamic changes throughout (all P > 0.10), as well as metaraminol (P = 0.49) and inotrope requirements (P > 0.10), intraoperative myocardial ischemia (P = 0.12) and perioperative myocardial infarction (P = 0.50). The results of this trial suggest that a propofol-based anesthetic, when compared to an enflurane-based anesthetic requiring additional dosing of fentanyl and midazolam for CPB, can lead to a significant reduction in time to extubation after CABG surgery, without adverse hemodynamic effects, increased risk of myocardial ischemia or infarction.

Objective: To document changes in serum lipids and glucose with a propofol infusion technique for cardiac surgery., Design: Prospective cohort., Setting: University teaching hospital., Participants: 22 elective cardiac surgical patients., Interventions: Frequent venous blood sampling., Measurements and Main Results: Serum lipids and glucose were measured at 10 time periods perioperatively, from preinduction until 4 hours post-cardiopulmonary bypass. Plasma propofol concentrations were also measured in 10 of these patients. There was a significant increase in glucose (P < 0.0005) and decreases in cholesterol (P < 0.0005), high-density lipoprotein (P = 0.004), and low-density lipoprotein (P < 0.0005); there was no significant change in triglycerides (P = 0.39). The propofol infusion resulted in acceptable plasma levels throughout the procedure and allowed early extubation in the intensive care unit, after a mean (SD) of 7.14 (5.9) hours. There was a strong correlation between triglyceride and propofol levels at most time periods (r = 0.38 to 0.98)., Conclusions: This study demonstrates that a propofol infusion technique does not result in elevation of serum lipids and supports its increased popularity in maintenance of anesthesia for cardiac surgery.