Thioester Linkage Research Articles

Dynamics in an unusual acyl carrier protein from a ladderane lipid-synthesizing organism.

Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual "ladderane" lipids these organisms produce. This "anammox-specific" ACP, or amxACP, shows several unique features such as a conserved FF motif and an unusual sequence in the functionally important helix III. Investigation of the protein's structure and dynamics, both in the crystal by ensemble refinement and by MD simulations, reveals that helix III adopts a rare six-residue-long 310 -helical conformation that confers a large degree of conformational and positional variability on this part of the protein. This way of introducing structural flexibility by using the inherent properties of 310 -helices appears unique among ACPs. Moreover, the structure suggests a role for the FF motif in shielding the thioester linkage between the protein's prosthetic group and its acyl cargo from hydrolysis.

Selected Articles from This Issue

Resistance to current lines of therap. in gastrointestinal stromal tumors (GIST) can occur through a variety of secondary mutations that occur in KIT. Novel therapeutics are still needed to address the mutational heterogeneity of KIT in GIST patients. In this study, Gupta and collaborators demonstrate the broad spectrum of ripretinib against primary and secondary drug-resistant mutations. Ripretinib is a “switch-control” kinase inhibitor designed to address even aggressively activated mutants to generate inactive conformations. Knowing that imatinib and MEK inhibition previously showed promise against imatinib-sensitive tumors, the authors also demonstrate the potency of combining ripretinib with MEKi in vitro and in vivo.KRAS mutations are present in up to 95% of pancreatic ductal adenocarcinoma (PDAC) cases. Inhibiting MEK (downstream of KRAS) would therefore appear to be a viable strategy for treating PDAC. Unfortunately, MEK inhibition as a monotherapy has been suboptimal in preclinical and clinical studies. Since CDKN2A mutations are also prevalent in a majority of PDAC tumors, Willobee and collaborators investigated the combination of CDK4/6 inhibitors with MEK inhibition. Combined CDK4/6 and MEK inhibition resulted in a significant survival benefit in an aggressive genetic mouse model of PDAC and provided a mechanistic rationale to explore the combination in clinical studies.For many cancers, the overexpression of P-cadherin is significantly correlated with lower overall and progression-free survival. Due to its limited expression in normal tissue and broad expression in tumors, Sheng and colleagues developed the P-cadherin specific antibody-drug conjugate PCA062. PCA062 contains a P-cadherin specific antibody bound to DM1 via a non-cleavable thioester linkage SMCC. The in vivo efficacy of PCA062 was demonstrated in cell- and patient-derived xenograft models of esophageal, triple-negative breast, and head and neck cancers. The authors note that limited efficacy signals were observed at dose escalation in Phase 1 trials. Taken together, the authors narrate the promise and challenges in targeting P-cadherin with the ADC modality.Inhibition of the programmed cell death ligand 1 (PD-1) axis has succeeded in many different solid tumors. The response of glioblastoma to PD-1 inhibition, however, remains limited due to its cold immune environment. Zhang and colleagues analyzed data from TCGA and clinical glioma samples and determined that Wnt/β-catenin expression was inversely related to the degree of immune cell infiltration and PD-L1 expression. They demonstrate that Wnt inhibition enhanced anti-PD-1 antibodies in a homograft mouse model by increasing lymphocyte infiltration and IFN-γ secretion. Their results demonstrate the potential for modifying the glioblastoma tumor microenvironment using Wnt inhibitors to potentiate immune checkpoint inhibition.

Mechanistic Investigation of ε-Thiono-Caprolactone Radical Polymerization: An Interesting Tool to Insert Weak Bonds into Poly(vinyl esters)

The incorporation of heteroatoms into the backbone of commodity polymers prepared by radical polymerization is an elegant way to confer (bio)degradability to such materials. This could be achieved via the radical ring-opening polymerization of cyclic monomers. Thionolactones were recently identified as promising comonomers for rROP but only reacting with activated monomers such as acrylate and acrylamide derivatives. Herein, we describe two thionolactone monomers, oxepane-2-thione i.e. ethiono-caprolactone (thCL) and 7-butyloxepane-2-thione, i.e e-thiono-decalactone (thDL) capable of performing radical ringopening polymerization with less activated monomers such as vinyl acetate (VAc) to produce readily degradable copolymers via the insertion of thioester containing repeating units. A thorough mechanistic investigation was performed to understand the reactivity of the two cyclic monomers. We identified the initiation as the main parameter to perform the polymerization of such thionolactone monomers. In a specific case the poly(e-thCL) homopolymer was successfully obtained. Concerning the copolymerization with vinyl acetate, a variety of polymerizations with differing feed ratios were performed, and the degradability of the copolymers via aminolysis examined by SEC. To demonstrate the usefulness of thionolactones a variety of block copolymers containing thioester linkages involving N,N-dimethylacrylamide and VAc were also prepared and degraded.

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications.

Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages. In this review, the specific click chemical reactions that have been employed to study protein lipid modification and their use for specific labeling applications are first described. This is followed by an introduction to the different types of protein lipid modifications that occur in biology. Next, the roles of click chemistry in elucidating specific biological features including the identification of lipid-modified proteins, studies of their regulation, and their role in diseases are presented. A description of the use of protein-lipid modifying enzymes for specific labeling applications including protein immobilization, fluorescent labeling, nanostructure assembly, and the construction of protein-drug conjugates is presented next. Concluding remarks and future directions are presented in the final section.

In a mouse model of INCL reduced S‐palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation

S-palmitoylation is a reversible posttranslational modification in which a 16-carbon saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. Dynamic S-palmitoylation (palmitoylation-depalmitoylation), like phosphorylation-dephosphorylation, regulates the function of numerous proteins, especially in the brain. While a family of 23 palmitoyl-acyl transferases (PATS), commonly known as ZDHHCs, catalyze S-palmitoylation of proteins, the thioesterases, localized either in the cytoplasm (eg, APT1) or in the lysosome (eg, PPT1) mediate depalmitoylation. Previously, we reported that APT1 requires dynamic S-palmitoylation for shuttling between the cytosol and the plasma membrane. APT1 depalmitoylated H-Ras to regulate its signaling pathway that stimulates cell proliferation. Although we demonstrated that APT1 catalyzed its own depalmitoylation, the ZDHHC(s) that S-palmitoylated APT1 had remained unidentified. We report here that ZDHHC5 and ZDHHC23 catalyze APT1 S-palmitoylation. Intriguingly, lysosomal Ppt1-deficiency in Cln1-/- mouse, a reliable animal model of INCL, markedly reduced ZDHHC5 and ZDHHC23 levels. Remarkably, in the brain of these mice decreased ZDHHC5 and ZDHHC23 levels suppressed membrane-bound APT1, thereby, increasing plasma membrane-localized H-Ras, which activated its signaling pathway stimulating microglia proliferation. Increased inflammatory cytokines produced by microglia together with increased complement C1q level contributed to the transformation of astrocytes to neurotoxic A1 phenotype. Importantly, neuroinflammation was ameliorated by treatment of Cln1-/- mice with a PPT1-mimetic small molecule, N-tert(Butyl)hydroxylamine (NtBuHA). Our results revealed a novel pathway to neuropathology in an INCL mouse model and uncovered a previously unrecognized mechanism of the neuroprotective actions of NtBuHA and its potential as a drug target.

Herpes simplex virus 1 infection induces ubiquitination of UBE1a.

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

On-demand retrieval of cells three-dimensionally seeded in injectable thioester-based hydrogels.

Scaffold systems that can easily encapsulate cells and safely retrieve them at the desired time are important for the advancement of cell-based medicine. In this study, we designed and fabricated thioester-based poly(ethylene glycol) (PEG) hydrogels with injectability and on-demand degradability as new scaffold materials for cells. Hydrogels can be formed in situ within minutes via thioester cross-linking between PEG molecules and can be degraded under mild conditions in response to l-cysteine molecules through thiol exchange occurring at the thioester linkage. Various cell experiments, especially with sucrose, which enables the adjustment of the osmotic pressure around the cells, showed that the damage to the cells during encapsulation and degradation was minimal, indicating the capability of on-demand retrieval of intact cells. This hydrogel system is a versatile tool in the field of cell-based research and applications such as tissue regeneration and regenerative medicine.

ε-Thionocaprolactone: an accessible monomer for preparation of degradable poly(vinyl esters) by radical ring-opening polymerization

Readily accessible ε-thionocaprolactone can be copolymerized with vinyl esters under radical polymerization conditions to produce copolymers containing degradable thioester and thioacetal linkages.

S-palmitoylation of NOD2 controls its localization to the plasma membrane

Nucleotide-binding and oligomerization domain containing protein 2 (NOD2) is a cytosolic pattern-recognition receptor that detects intracellular peptidoglycan (muramyl dipeptide) from bacteria. Membrane association of NOD2 is essential for its ability to activate nuclear factor κB and mitogen-activated protein kinase signaling pathways via the kinase, receptor-interacting serine/threonine-protein kinase 2. The post-translational addition of palmitate to NOD2 results in an acylated protein with increased affinity for membrane bilayers (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. et al.Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (60) Google Scholar). Palmitoylation of cysteine residues (shown in orange) at positions C395 and C1033 en face of the structural model (A) based on the crystal structure of rabbit NOD2 (PDB ID: 5IRL) (2Maekawa S. Ohto U. Shibata T. Miyake K. Shimizu T. Crystal structure of NOD2 and its implications in human disease.Nat. Commun. 2016; 7: 11813Crossref PubMed Scopus (102) Google Scholar) is mediated by the protein acyltransferase enzyme zDHHC5 (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. et al.Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (60) Google Scholar). The model suggests that this face of the protein is juxtaposed to the plasmalemmal surface. Palmitoylation of the expressed GFP-NOD2WT and GFP-NOD2C395,1033S in HCT116 cells was further characterized using a biorthogonal chemical reporter assay (B) (3Yap M.C. Kostiuk M.A. Martin D.D.O. Perinpanayagam M.A. Hak P.G. Siddam A. Majjigapu J.R. Rajaiah G. Keller B.O. Prescher J.A. Wu P. Bertozzi C.R. Falck J.R. Berthiaume L.G. Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry.J. Lipid Res. 2010; 51: 1566-1580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Here, 15-hexadecynoic acid (15-HDYA), also referred to as alkynyl palmitic acid, was metabolically incorporated into cells. The 15-HDYA covalently attached to the immunocaptured GFP-NOD2 proteins was reacted with azide-PEG3-FLAG via a copper-catalyzed azide–alkyne cycloaddition reaction. The proteins were resolved by SDS-PAGE and subsequently detected by an anti-FLAG antibody. The metabolic label, which was incorporated into NOD2WT and was notably absent in the double cysteine mutant, was removed by treatment with 2.5% hydroxylamine that hydrolyzes the 15-DYA–protein thioester linkage. Finally, the palmitoylation-deficient GFP-NOD2C395,1033S does not localize to the plasma membrane in HCT116 cells (C). Mutations that disrupt S-palmitoylation are associated with severe immunologic and inflammatory diseases such as Crohn’s disease, whereas mutations associated with Blau syndrome demonstrate increased S-palmitoylation and membrane localization (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. et al.Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (60) Google Scholar). EQUIPMENT: The following equipment were used: A Zeiss Axiovert 200M microscope with a Hamamatsu ImageEM x2 camera, Yokogawa spinning disc, and a 63× 1.4 NA oil objective. REAGENTS: Alkynyl-palmitate (Click Chemistry Tools), azide-PEG3-FLAG (Jena Bioscience), hydroxylamine, and other copper-catalyzed azide–alkyne cycloaddition reagents (TCEP, CuSO4, and TBTA) were obtained from MilliporeSigma. SOFTWARE: The following software were used: MetaMorph, ImageJ, Python, PyMOL, and AutoDock (4Trott O. Olson A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.J. Comput. Chem. 2009; 31: 455-461Google Scholar).

Protein palmitoylation and its pathophysiological relevance.

Protein palmitoylation, in which C16 fatty acid chains are attached to cysteine residues via a reversible thioester linkage, is one of the most common lipid modifications and plays important roles in regulating protein stability, subcellular localization, membrane trafficking, interactions with effector proteins, enzymatic activity, and a variety of other cellular processes. Moreover, the unique reversibility of palmitoylation allows proteins to be rapidly shuttled between biological membranes and cytoplasmic substrates in a process usually controlled by a member of the DHHC family of protein palmitoyl transferases (PATs). Notably, mutations in PATs are closely related to a variety of human diseases, such as cancer, neurological disorders, and immune deficiency conditions. In addition to PATs, intracellular palmitoylation dynamics are also regulated by the interplay between distinct posttranslational modifications, including ubiquitination and phosphorylation. Understanding the specific mechanisms of palmitoylation may reveal novel potential therapeutic targets for many human diseases.

Dynamic palmitoylation events following T-cell receptor signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation.

Geminal Dimethyl Substitution Enables Controlled Polymerization of Penicillamine-Derived β-Thiolactones and Reversed Depolymerization

Summary To access infinitely recyclable plastics, one appealing approach is to design thermodynamically neutral systems based on dynamic covalent bond, the (de)polymerization of which can be easily manipulated with low energy cost. Here, we demonstrate the feasibility of this concept via the efficient synthesis of polythioesters PNR-PenTE from penicillamine-derived β-thiolactones and their convenient depolymerization under mild conditions. The gem-dimethyl group adjusts the thermodynamics of (de)polymerization to near equilibrium, confers better (de)polymerization control by reducing the activity and conformational possibilities of the chain-end thiolate groups, and stabilizes the thioester linkages in the polymer backbone. PNR-PenTE with tailored properties is conveniently accessible by altering the side chains. PNR-PenTE can be recycled to pristine enantiopure β-thiolactones at >95% conversion from minutes to a few hours at room temperature. This work highlights the power of judicious molecular design and could greatly facilitate the development of a wide range of recyclable polymers with immense application potentials.

Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1-2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.

Development of 6-Thioguanine conjugated PLGA nanoparticles through thioester bond formation: Benefits of electrospray mediated drug encapsulation and sustained release in cancer therapeutic applications.

Polymeric nanoparticle-based successful delivery of hydrophobic drugs is highly desirable for its controlled and sustained release at the disease site, which is a challenge with the current synthesis methods. In the present study, an electrospray mediated facile one-step synthesis approach is explored in which a solution mixture of a hydrophobic drug, 6-thioguanine (Tg) and a biocompatible FDA approved polymer, Poly (d, l-lactide-co-glycolide) (PLGA) is injected in an applied electric field of suitable intensity to prepare drug encapsulated PLGA nanoparticles, PLGA-Tg with high yield. In order to explore the effect of external electric field on Tg loading and delivery applications, the nanoparticles are characterized using EDX, AFM, FESEM, TEM, FTIR, Raman, fluorescence, and mass spectroscopy techniques. The characterization studies indicate that the electric field mediated synthesis exhibits spherical nanoparticles with a homogenous core size distribution of ~60nm, high encapsulation (~97.22%) and stable conjugation of Tg (via thioester linkages) with PLGA molecules in the presence of the applied electric field. The kinetic study demonstrates the 'anomalous diffusion' (non-Fickian diffusion) release mechanism in which Tg escapes from PLGA matrix with a slow, but steady diffusion rate and the sustained drug release profile continues for 60days. To check the biological activity of the encapsulated Tg, in-vitro cell studies of the PLGA-Tg are performed on HeLa cells. The MTT assay shows significant cell death after 48h of treatment, and the cellular internalization of the drug-loaded nanoparticles occurs through pinocytosis mediated uptake, which is established by the AFM analysis. The Raman and mass spectroscopy studies suggest that the PLGA-Tg nanoparticles are rapidly hydrolyzed inside cell cytoplasm to release Tg which initiates apoptosis-mediated cell death confirmed by as DNA fragmentation and membrane blebbing studies. The results clearly emphasize the benefits of electrospray based synthesis of polymeric nanodrug formulation through the formation of chemical bonds between polymer and drug molecules that could be easily implemented in the design and development of an effective nanotherapeutic platform with no typical 'burst effect,' prolonged release profile, and significant toxicity to the cancer cells.

Investigation of Post‐Translational Modification on NHE1 Transport Function

The sodium hydrogen exchanger isoform 1 (NHE‐1) is a sodium hydrogen antiporter that regulates intracellular pH (pHi), cell volume, dynamic actin remodeling processes, and coordination of cell migration in mammalian tissues. NHE‐1 is post‐translationally modified by phosphorylation and lipid modification (palmitoylation). The intracellular C‐terminal domain of NHE‐1 is reversibly phosphorylated at multiple sites byseveral kinases including: extracellular signal‐regulated kinase (ERK), protein kinase B (AKT), B‐Raf, p90 Ribosomal S6 Kinase (p90rsk), RhoA Kinase (ROCK), and other protein kinases. We recently identified that NHE‐1 is reversibly palmitoylated (S‐acetylation), in which a 16‐carbon palmitic acid is covalently attached to a cysteine residue on the C‐terminal domain via a thioester linkage. Such modifications are reported to regulate protein trafficking, membrane micro localization, and protein‐protein interactions. Steady‐state pHi assays incubated with pH‐sensitive dye were conducted to measure the change in pHi of Chinese Hamster Lung Fibroblast Cells due to NHE‐1. Investigating agonists and stimulating factors that increase NHE‐1 palmitoylation allows us to determine the impact of palmitoylation on NHE‐1 transport. Palmitoyl‐transferases (PATs) catalyze the lipidation of proteins and are inhibited by 2‐bromo‐palmitate (2BP). The presence of 2BP causes a loss of palmitoylation to occur, effectively inhibiting LPA‐induced NHE‐1 activity by 0.1 pH units. Fetal Bovine Serum (FBS), Lysophosphatidic acid (LPA), Phorbol 12‐myristate 13‐acetate (PMA), and Insulin effectively stimulate NHE‐1 by increasing pHi by 0.1 pH units. In the presence of kinase inhibitors, NHE‐1 activity significantly decreases compared to agonist or serum treatment alone. The impact of the combination of kinase inhibitors and palmitoylation inhibitors on NHE‐1 mediated pHi change was determined. The data supports the hypothesis that palmitoylation and phosphorylation coordinate to subtly modify NHE1 activity in a novel rheostat of the cell.

DHHC21‐mediated α1 Adrenergic Receptor Palmitoylation Contributes to Renal Blood Flow Regulation During Sepsis

Palmitoylation is a reaction of 16‐carbon palmitic acid attachment to a cysteine residue of a protein via thioester linkage, catalyzed by a family of palmitoyl acyltransferases containing the DHHC motif. This post‐translational modification plays a critical role in regulating protein function, subcellular localization and interactions with other cellular molecules. A recent study shows that vascular α1 adrenergic receptor (α1AR) requires DHHC21‐mediated palmitoylation for its signaling activity to induce vasoconstriction. In the kidney, arterial vasoconstriction has been implicated in the development of renal hypoperfusion following sepsis. In this study, we sought to examine whether DHHC21 contributes to kidney dysfunction by affecting renal blood flow through α1AR palmitoylation. Cecal ligation puncture (CLP) was performed to induce polymicrobial sepsis in wild type and DHHC21 functional deficient (Zdhhc21dep/dep) mice. Their renal function was dynamically assessed based on tracer (IRdye 800CW) clearance measured by multispectral optoacoustic tomography (MSOT). The results showed that in wild‐type mice, IRdye 800CW movement from kidney cortex to the medulla/pelvis region was significantly slower after CLP, compared to sham control. The sepsis‐induced impairment in renal clearance was alleviated in Zdhhc21dep/dep mice. Consistently, histological analysis (Periodic acid‐Schiff staining) revealed that CLP caused kidney structural damages, evidenced by tubular vacuolation, brush border loss, tubular cell detachment and leukocyte infiltration; these pathologies were greatly ameliorated in DHHC21 loss‐of‐function mice. Moreover, compared with wild‐type mice, Zdhhc21dep/dep mice exhibited reduced CLP‐induced increases in circulating creatinine and urea nitrogen. Mechanistically, a decreased ratio of oxygenated to deoxygenated hemoglobin was detected in kidneys of wild‐type mice following CLP, suggesting renal tissue hypoxia in response to sepsis. We then asked whether this lack of oxygenation is associated with reduced renal blood flow due to arterial constriction and whether α1AR palmitoylation is involved in this process. Indeed, we found a significant increase in α1AR palmitoylation, along with activation of the α1AR downstream effector ERK, in renal arteries of wild‐type mice following CLP. These molecular responses occurred concomitantly with a reduced renal blood flow, measured by a transonic flowmeter. In contrast, sepsis‐induced α1AR palmitoylation/activation in renal arteries was greatly attenuated in Zdhhc21dep/dep mice, consistent with a better‐preserved renal blood flow in these mice. Taken together, our findings indicate that DHHC21‐mediated α1AR palmitoylation contributes to renal blood flow reduction and kidney injury during sepsis.Support or Funding InformationThis work was supported by the National Institutes of Health Grant HL070752 (to SYY)

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.

Enabling Combinatorial siRNA Delivery against Apoptosis-Related Proteins with Linoleic Acid and α-Linoleic Acid Substituted Low Molecular Weight Polyethylenimines

Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. The effective polymers formed 80-190nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2kDa and 2.0kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.

A Computational Study of the Role of the E3 Ligase in the Ubiquitination Reaction

Post translational modifications can change protein functions without changing the entire protein. Ubiquitination is a type of post translation modification wherein the small protein Ubiquitin (Ub) is covalently bound to a lysine on a target protein. One well-known function of ubiquitination is to flag proteins to be degraded. Ubiquitination occurs by a multitude of reactions catalyzed by three types of enzymes: ubiquitin activating enzymes, E1; ubiquitin conjugating enzymes, E2; and ubiquitin ligases, E3. The E2 enzyme directly catalyzes the ubiquitin transfer, whereas the E3 enzyme improves the efficiency of ubiquitination but it does not affect the chemical mechanism. In the accepted mechanism of the E2 enzyme, Ub is covalently linked to the E2 via a thioester bond and ubiquitination proceeds through a zwitterionic, tetrahedral intermediate. It is suggested that the negative charge on the zwitterion is stabilized by a hydrogen bond to a nearby, highly conserved asparagine (Asn79 in Ubc13). It is hypothesized that the E3 enzyme improves the catalytic efficiency of ubiquitination by placing the E2∼Ub conjugate in a “closed” position, which may put bond strain on the thioester linkage. We have analyzed how the hydrogen bond environment changes in the open and closed E2∼Ub positions by running molecular dynamics simulations starting from protein databank (PDB) crystal structures 5ait (closed) and 2gmi (open). Our data shows that systems in the closed position increase hydrogen bonding to the substrate. We have also used density functional theory on a model reaction to show that a modest amount of bond strain can result in improved catalysis.

Regulation of NCX1 by palmitoylation

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.