Lysine Analogues Research Articles

Comparative efficacy of tranexamic acid versus placebo on hemostatic outcomes after the surgical debridement of burn patients: A randomized controlled clinical trial

IntroductionBurns are traumatic events that can affect multiple systems beyond the skin. The rapid removal of the burn eschar is a key step in the effective treatment of severe burns, and surgical debridement is currently the standard of care for eschar removal in burn patients. However, surgical debridement is highly hemorrhagic. Tranexamic acid, an antifibrinolytic lysine analog, may reduce bleeding. MethodsA randomized control trial was conducted to examine the effects of preoperative tranexamic acid administration in the early surgical debridement of severe burns. The study was conducted from January to December 2022. ResultsThe general characteristics of patients and mechanism of injury were similar between the treated and control groups. The burned body surface area was 26.46% ± 5.45% in the study group and 27.10% ± 4.45% in the control group (p = 0.83). Intraoperative hemorrhage volume was larger in the control group (299.3 ± 88.9ml) than in the study group (117 ± 51.7ml) (p = 0.0001). The decreases in hemoglobin and hematocrit levels were larger in the control group (2.1 ± 1.4g/dl and 5.7% ± 4.6%) than in the study group (0.85 ± 0.4g/dl and 2.1% ± 0.5%) (p = 0.004 and 0.01, respectively). Six patients in the control group but no patients in the study group required blood transfusion (p = 0.002). No thromboembolic events were observed. ConclusionThese findings show that tranexamic acid was effective at reducing intraoperative hemorrhage volume and the need for transfusion in burn surgery patients. It was shown to be safe for use in these patients.

Tranexamic Acid for reduction of intra- and postoperative TRansfusion requirements in elective Abdominal surgery (TATRA): study protocol for an investigator-initiated, multicenter, double-blind, placebo-controlled, randomized superiority trial with two parallel groups

BackgroundIntra- and postoperative hemorrhage is a relevant problem in major abdominal surgery, leading to acute anemia and necessitating transfusion of packed red blood cells. It is estimated that in 30% of abdominal surgeries, intra- or postoperative transfusion is required. Transfusion potentially has detrimental health effects and poses a considerable socioeconomic burden. Tranexamic acid, a lysine analog inhibiting plasminogen activation and providing clot stability, has been used to reduce hemorrhage. While there is ample evidence in other surgical disciplines, it is almost completely lacking in abdominal surgery.MethodsThis multicenter double-blind parallel group randomized superiority trial will compare tranexamic acid (loading dose 1000 mg over 10 min prior to skin incision, maintenance dose 125 mg/h continuously until skin closure or until 1000 mg have been administered) to placebo in patients ≥ 18 years undergoing elective esophagectomy, gastrectomy, colectomy, rectal resection, pancreatic resection, or hepatectomy. The primary efficacy endpoint is the intra- or postoperative transfusion of at least one unit of packed red blood cells. Key secondary endpoints are the number of transfused units per patient, estimated intraoperative blood loss, postoperative complications/mortality, length of hospital stay, operation/anesthesia time, D-dimer levels, and quality of life. Sample size calculation is based on the assumption that in the control group, 30% of patients require transfusion while the intervention achieves a risk reduction of 33%, reducing the probability to 20%. With a type one error of 5% and a power of 90%, using a two-sided χ2 test, this results in 412 patients per group. Accounting for non-compliance, 425 patients are to be randomized per group. The total trial duration will be 30 months with a recruitment period of 18 months.DiscussionIf the proposed trial yielded positive results, the routine use of tranexamic acid in major abdominal surgery would be supported. This would avoid acute anemia with detrimental effects such as tissue hypoxia and organ injury, as well as the negative immediate and delayed effects of transfusions.Trial registrationEU CT Nr: 2023–509970-43–01, NCT06414031. Registered on 10 May 2024.

Effectiveness of tranexamic acid on intra- and postoperative bleeding in Bimaxillary osteotomies: a retrospective study.

Perioperative bleeding is a serious concern during orthognathic surgery. Tranexamic acid (TXA), a synthetic lysine analog with antifibrinolytic properties, reduces blood loss across various surgical fields. This study aimed to investigate the effectiveness of preoperative TXA administration in reducing intraoperative and postoperative blood loss following combined Le Fort I and sagittal split ramus osteotomies at our hospital. This single-center, retrospective cohort study included patients who underwent combined Le Fort I and sagittal split ramus osteotomies between November 2017 and October 2022. The primary outcome was the volume of intraoperative blood loss. Among 1,329 eligible patients, 87 were included in the analysis (32 in the TXA group and 55 in the control group, where no TXA was administered). The median (interquartile range) intraoperative blood loss was 200.0 (157.5-237.5) mL in the TXA group and 260.0 (180.0-350.0) mL in the control group, showing a significant difference between the groups (p = 0.0365). However, postoperative blood drainage within 24h and 24-48h did not differ significantly between the two groups. A single intravenous administration of TXA was associated with a decrease in intraoperative bleeding without severe adverse events during combined Le Fort I and sagittal split ramus osteotomies. However, postoperative blood loss, nausea, vomiting, and autologous blood transfusion were not significantly associated with this administration.

Comparative Study of Topical Application of Injection Tranexamic Acid and Anterior Nasal Packing in the Management of Epistaxis.

Epistaxis, defined as bleeding from the nose, is one of the common ENT cases coming to emergency department. Epistaxis is experienced by at least 60% of the population once in their life time and about 6% of them will require medical attention. The different treatment modalities include: local pressure, application of topical vasoconstrictor substances, or nasal packing depending on personal physician preference. Tranexamic acid (TXA), a synthetic analogue of the amino acid lysine, belongs to a class of drugs known as antifibrinolytics. It acts by reversibly binding four to five lysine receptor sites on plasminogen and can be used in emergency department for reducing the bleeding time in epistaxis. To evaluate the efficacy of topical application of injection TXA compared to cases managed with anterior nasal packing for the treatment of patients with epistaxis. 100 patients presenting with epistaxis in emergency department, above the age of 18years were randomly divided into two groups with 50 patients each. Group 1 were managed with anterior nasal packing with gel foam and Group 2 with topical application of injection TXA. Causes,duration to control epistaxis, and occurrence of rebleeding were recorded. Our study showed homogenous distribution of age and sex among the patients. Bleeding stopped within 10min in 38 patients in group 2 compared to 17 patients in group 1. For 31 patients in group 1, bleeding stopped between 10 and 15min compared to 12 in group 2. In group 1, 8 patients had rebleeding compared to 2 patients in group 2. Our study showed that topical application of TXA reduces the bleeding time and number of rebleeds compared to anterior nasal packing with gelfoam. Since it is easily available in an emergency setup and cheaper compared to gelfoam, it can be used as an elective method in managing epistaxis in emergency department.

The effects of tranexamic acid in patients treated with extracorporeal membrane oxygenation after cardiac surgery.

The lysine analog tranexamic acid (TXA) is used as a blood protective drug in cardiac surgery, but efficacy and safety outcomes in patients treated with extracorporeal membrane oxygenation (ECMO) after surgery remain poorly understood. From January 1, 2017 to December 31, 2022, we retrospectively analyzed patients assisted by ECMO after cardiac surgery and divided them into TXA and control groups depending on whether TXA was used or not. The primary study outcome was red blood cell (RBC) transfusion during ECMO. In total, 321 patients treated with ECMO after cardiac surgery were assessed; 185 patients were eligible for inclusion into to the TXA-intervention group and 136 into to the control group. RBC transfusion during ECMO was 8.0 IU (4.0 IU-14.0 IU) in the TXA group versus 10.0 IU (6.0 IU-16.0IU) in the control group (p = .034). Median total chest drainage volume after surgery was 1460.0mL (650.0-2910.0mL) and 1680.0mL (900.0-3340.0mL) in TXA and control groups, respectively (p = .021). Postoperative serum D-dimer levels were significantly lower in the TXA group when compared with the control group; 1.125µg/mL (0.515-2.176µg/mL) versus 3.000µg/mL (1.269-5.862µg/mL), p < .001. Serious adverse events, including vascular occlusive events, did not differ meaningfully between groups. In patients treated with ECMO after cardiac surgery, TXA infusion modestly but significantly reduced RBC transfusions and chest tube output when compared with the control group.

Genetically Encoded Lysine Analogues with Differential Light Sensitivity for Activation of Protein Function

AbstractGenetically encoded unnatural amino acids are versatile tools for controlling protein function, but options for regulating multiple proteins in a single experiment are limited. Here, we report the genetic encoding of two new photocaged lysine derivatives, 1‐(2‐nitrophenyl)‐ethyl lysine and nitrodibenzylfuranyl lysine, for sequential light‐activation of protein function in live cells. Nitrodibenzylfuranyl (NDBF) caging groups have a redshifted absorbance maximum and high sensitivity to light compared to the 1‐(2‐nitrophenyl)‐ethyl group (NPE), enabling selective decaging and protein activation. We characterized the responses of these new caged amino acids by optically triggering nuclear localization and firefly luciferase activity. The ability to selectively activate distinct proteins through simple light titration makes this a useful approach with broad applications.

Safety and efficacy of tranexamic acid in major non-cardiac vascular surgery: a systematic review and meta-analysis

Background: Tranexamic acid (TXA) is a synthetic lysine analogue that inhibits fibrinolysis. The effectiveness of TXA in obstetrics, trauma and orthopaedic and cardiac surgery is well established. However, concerns regarding its prothrombotic potential remain, which is an important consideration for vascular surgery. We aimed to evaluate the safety and efficacy of TXA in adults undergoing major non-cardiac vascular surgery. Methods: A pre-specified protocol (PROSPERO CRD42023427282) was followed. Relevant databases (PubMed, MEDLINE, EMBASE) were searched for randomised controlled trials (RCTs). Data extraction and risk of bias assessments were performed in duplicate. A random effects model was used to synthesise data from RCTs. Measures of effect were reported as relative risk (RR) with 95% confidence intervals (CIs). The primary safety outcome was a composite of arterial and venous thromboembolic events (composite of myocardial infarction, non-haemorrhagic stroke, peripheral arterial thrombosis and symptomatic proximal venous thromboembolism). Certainty of evidence was assessed using the GRADE approach. Results: After screening 1989 records, three RCTs were included, cumulatively enrolling 1,560 participants. In all trials, patients received either TXA (intravenously or orally) compared with placebo. There was no evidence of the effect of intravenous TXA on thromboembolic events (RR 1.10, 95% CI 0.89 to 1.36, two RCTs, 1460 participants, low certainty of evidence) or on critical bleeding (composite of life-threatening, critical and major organ bleed) (RR 0.85, 95% CI 0.65 to 1.11, two RCTs, 1460 participants, low certainty of evidence). TXA may reduce postoperative blood loss, especially at 0–4 hours (Cliff’s delta −0.41, 95% CI −0.19 to −0.59) and 0–24 hours (Cliff’s delta −0.37, 95% CI −0.14 to −0.55) after surgery. There was no evidence of an effect of TXA on reducing perioperative red blood cell (RBC) transfusion requirements (RR 0.66, 95% CI 0.11 to 3.95, one RCT, 100 participants). One RCT assessed oral TXA and found no evidence of an effect on type II endoleak post endovascular aneurysm repair. No thrombotic complications were reported in this RCT during the study period. Conclusions: We found no evidence of an effect of TXA on thromboembolic complications. While TXA appears to reduce early postoperative bleeding, the clinical relevance of this is uncertain. Due to limitations of study design and the variety of vascular procedures, the role of TXA in vascular surgery is still unclear. Ongoing trials may reduce this uncertainty. Registration: Prospectively registered on PROSPERO (CRD42023427282)

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

Tranexamic acid (class I drug) reduced and capped gold nanoparticles as a potential hemostatic agent with enhanced performance

External hemostatic agents play a crucial role in stabilizing an impaired process during pathological conditions. The idea is to stabilize the in vivo system as soon as possible. This study uses a class I hemostatic drug tranexamic acid as a reducing and capping agent for synthesizing the gold nanoparticles (Tr-AuNPs). Being the synthetic analogue of lysine and a biologically inspired alkylamine molecule, the chemistry can be fine-tuned for stable material that can simultaneously target the intrinsic and extrinsic hemostatic pathway, making it promising for hemostatic applications. The Tr-AuNPs of hydrodynamic diameter ∼46 nm were synthesized and evaluated physio-chemically using various analytical techniques wherein they showed hemocompatibility and increased thrombus weight compared to the native drug. The decrease in prothrombin time (PT) and international normalized ratio supported by the dynamic thromboelastography (TEG) study indicates the prepared nano-conjugate’s potential in reducing time for attaining hemostasis as compared to the native tranexamic acid drug. At a 9 μg ml−1 concentration, Tr-AuNPs had a procoagulant effect, shown by decreased reaction time (R) and coagulation time (K) with improved α angle and MA. There was a significant increase in the rate of coagulation in vivo by Tr-AuNPs, i.e. (52 s) compared to the native tranexamic acid (360 s). Radiolabelling studies ascertained the in vivo biocompatibility (non-invasive distribution, residence, clearance, and stability) of the Tr-AuNPs. The short-term toxicity studies were conducted to establish a proof of concept for the biomedical application of the material. The results highlighted the use of biologically alkyl amine molecules as capping and reducing agents for the synthesis of nanoparticles, which have shown a synergistic effect on the coagulation cascade while holding the potential for also acting as potential theranostic agents.

Effect of Spacer Length Modification of the Cationic Side Chain on the Energetics of Antimicrobial Peptide Binding to Membrane-Mimetic Bilayers.

Understanding the mechanism of action of the antimicrobial peptide (AMP) in terms of its structure and energetics is the key to designing new potent and selective AMPs. Recently, we reported a membranolytic 14-residue-long lysine-rich cationic antimicrobial peptide (LL-14: NH3+-LKWLKKLLKWLKKL-CONH2) against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which is limited by cytotoxicity and expected to undergo facile protease degradation. Aliphatic side-chain-length modification of the cationic amino-acid residues (Lys and Arg) is a popular strategy for designing protease-resistant AMPs. However, the effect of the peptide side-chain length modifications on the membrane binding affinity and its relation to the atomic structure remain an unsolved problem. We report computer simulations that quantitatively calculated the difference in peptide binding affinity to membrane-mimetic-bilayer models (bacterial: 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) bilayer and mammalian: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer) upon decreasing or increasing the spacer length of the cationic lysine residues of LL-14 (as well as their arginine analogues). We show that the peptide/bilayer interaction energetics varies drastically in response to spacer length modification. The strength of peptide discrimination depends strongly on the nature of the bilayer (bacterial or mammalian mimetic model). An increase in the lysine spacer length by one carbon (i.e., homolysine analogue of LL-14) is weakly/strongly disfavored by the bacterial/mammalian-membrane-mimetic bilayer. Recently, we have demonstrated an excellent correlation between the antimicrobial activity of the membranolytic cationic peptides and their binding affinity to membrane-mimetic-bilayer models. Thus, the homolysine analogue of LL-14 is a promising noncytotoxic AMP with conserved activity. On the other hand, homoarginine analogue (arginine spacer length increment by a single carbon) was preferred by both the bacteria and the mammalian mimetic bilayers and displayed the strongest affinity for the former among the peptides studied in this work. Thus, the promising most potent homoarginine analogue is likely to be cytotoxic. Shortening the Lys/Arg side chain to a three-carbon spacer (Dab/Agb) improves the binding affinity to bacterial and mammalian-membrane-mimetic bilayers. Arginine and arginine-derivative peptides exhibited stronger binding affinity to the bilayers relative to the lysine analogue. The results provide a plausible explanation to the previous experimental observations, viz., superior antimicrobial activity of the arginine peptides relative to Lys peptides and the improvement of antimicrobial activity upon substitution of Lys with Dab in the cationic peptides. The simulations revealed that the small change in the peptide hydrophobicity by Lys/Arg spacer length modification could drastically alter the energetics of peptide/bilayer binding by fine-tuning the electrostatic interactions. The energetics underlying the peptide selectivity by simple membrane-mimetic bilayer models may be beneficial for designing new selective and protease-resistant AMPs.

Current Status of Tranexamic Acid and Antifibrinolytics

Tranexamic acid (TxA) is an antifibrinolytic used to reduce bleeding in many clinical settings, including cardiac surgery. It is a lysine analogue that acts by blocking the lysine-dependent production of plasmin and resultant fibrinolysis. This group has found that there is a broader role of the fibrinolytic system outside of haemostasis [ [1] Draxler DF, Yep K, Hanafi G, Winton A, Daglas M, Ho H, et al. Tranexamic acid modulates the immune response and reduces postsurgical infection rates. Blood Adv. 2019;3(10):1598–609. Google Scholar ]. Many components of the fibrinolytic system, including fibrin itself, have direct effects on immune function and inflammation, both stimulatory and inhibitory in nature.

C-Terminal Lysine Residue of Pneumococcal Triosephosphate Isomerase Contributes to Its Binding to Host Plasminogen

The main causative agent of pneumonia, Streptococcus pneumoniae, is also responsible for invasive diseases. S. pneumoniae recruits human plasminogen for the invasion and colonization of host tissues. We previously discovered that S. pneumoniae triosephosphate isomerase (TpiA), an enzyme involved in intracellular metabolism that is essential for survival, is released extracellularly to bind human plasminogen and facilitate its activation. Epsilon-aminocaproic acid, a lysine analogue, inhibits this binding, suggesting that the lysine residues in TpiA are involved in plasminogen binding. In this study, we generated site-directed mutant recombinants in which the lysine residue in TpiA was replaced with alanine and analyzed their binding activities to human plasminogen. Results from blot analysis, enzyme-linked immunosorbent assay, and surface plasmon resonance assay revealed that the lysine residue at the C-terminus of TpiA is primarily involved in binding to human plasminogen. Furthermore, we found that TpiA binding to plasminogen through its C-terminal lysine residue was required for the promotion of plasmin activation by activating factors.

Diagonal Interactions between Glutamate and Arginine Analogs with Varying Side-Chain Lengths in a β-Hairpin

Cross-strand interactions are important for the stability of β-sheet structures. Accordingly, cross-strand diagonal interactions between glutamate and arginine analogs with varying side-chain lengths were studied in a series of β-hairpin peptides. The peptides were analyzed by homonuclear two-dimensional nuclear magnetic resonance methods. The fraction folded population and folding free energy of the peptides were derived from the chemical shift data. The fraction folded population trends could be rationalized using the strand propensity of the constituting residues, which was not the case for the peptides with lysine analogs, highlighting the difference between the arginine analogs and lysine analogs. Double-mutant cycle analysis was used to derive the diagonal ion-pairing interaction energetics. The most stabilizing diagonal cross-strand interaction was between the shortest residues (i.e., Asp2-Agp9), most likely due to the least side-chain conformational penalty for ion-pair formation. The diagonal interaction energetics in this study involving the arginine analogs appears to be consistent with and extend beyond our understanding of diagonal ion-pairing interactions involving lysine analogs. The results should be useful for designing β-strand-containing molecules to affect biological processes such as amyloid formation and protein-protein interactions.

Abstract TP130: Effect Of Antifibrinolytic Medication On Hematoma Expansion In Acute Ischemic Stroke Patients With Post Thrombolytic Intracerebral Hemorrhages

Background and Purpose: Antifibrinolytic medications such as the synthetic lysine analogs tranexamic acid and ε-aminocaproic acid inhibit fibrinolysis by attaching to the lysine-binding site of the plasminogen molecule. However, the efficacy of intravenous (IV) antifibrinolytic medication in the prevention of hematoma expansion in acute ischemic stroke patients who develop post thrombolytic intracerebral hemorrhage (ICH) is unclear. Methods: We analyzed the effect of antifibrinolytic medication in acute ischemic stroke patients with post thrombolytic ICH on hematoma expansion over an 8-year period. The Region-of-Interest; method on a Horos Medical Image Viewer (Version 3.3.6) was utilized for hematoma volume measurement on serial computed tomographic scans. We compared changes in hemorrhage volume in patients who did and did not receive IV antifibrinolytic medication. Results: A total of 53 patients (mean age 69.7±14.3, 53% were men) with post thrombolytic ICH were analyzed. Twenty-six patients received antifibrinolytic treatment: 20 and 6 patients received a single IV dose of ε-aminocaproic acid (5 g) or tranexamic acid (1 g), respectively. Ten patients (38%) had a reduction or stabilization in hematoma volume (overall pre and post-treatment volumes were 17.43±16.23 cm3 and 25.14±26.57 cm3 respectively). Mean fibrinogen level in the antifibrinolytic medication group was 277 mg/dL; 3 patients had levels &lt;200 mg/dL. In patients that did not receive antifibrinolytics, 16 (59%) out of 27 patients had a reduction or stabilization in hematoma volume (overall pre and post-treatment volumes were 19.74±32.32 cm3 and 27.15±52.58 cm3 respectively). The mean percent change in hematoma volume with antifibrinolytic medication treatment was 48% and without was 27%. [Figure 1] Conclusion: We did not identify any reduction in any of the measures of hematoma expansion with antifibrinolytic treatment in acute ischemic stroke patients with post thrombolytic ICH.

Targeted protein degradation through light-activated E3 ligase recruitment.

Optical control of protein function through proteasomal degradation benefits from the noninvasive nature and spatiotemporal precision of light as a trigger. In this chapter, light activation of protein degradation with an optically controlled degron, termed optoDeg, is discussed. This method utilizes genetic code expansion to insert a photocaged analog of lysine at the N-terminal position of a protein of interest for spatial and temporal control of the N-end pathway, inducing proteasomal degradation. Methods for the use of optoDeg for degradation of the fluorescent reporter EGFP and the kinase MEK1 are described. The system is fast, with complete degradation of proteins within minutes following irradiation, and highly specific, with genetically directed introduction of the light-activated degron.

Lipidated Lysine and Fatty Acids Assemble into Protocellular Membranes to Assist Regioselective Peptide Formation: Correlation to the Natural Selection of Lysine over Nonproteinogenic Lower Analogues.

The self-assembly of prebiotically plausible amphiphiles (fatty acids) to form a bilayer membrane for compartmentalization is an important factor during protocellular evolution. Such fatty acid-based membranes assemble at relatively high concentrations, and they lack robust stability. We have demonstrated that a mixture of lipidated lysine (cationic) and prebiotic fatty acids (decanoic acid, anionic) can form protocellular membranes (amino acid-based membranes) at low concentrations via electrostatic, hydrogen bonding, and hydrophobic interactions. The formation of vesicular membranes was characterized by dynamic light scattering (DLS), pyrene and Nile Red partitioning, cryo-transmission electron microscopy (TEM) images, and glucose encapsulation studies. The lipidated nonproteinogenic analogues of lysine (Lys), such as ornithine (Orn) and 2,4-diaminobutyric acid (Dab), also form membranes with decanoate (DA). Time-dependent turbidimetric and 1H NMR studies suggested that the Lys-based membrane is more stable than the membranes prepared from nonproteinogenic lower analogues. The Lys-based membrane embeds a model acylating agent (aminoacyl-tRNA mimic) and facilitates the colocalization of substrates to support regioselective peptide formation via the α-amine of Lys. These membranes thereby assist peptide formation and control the positioning of the reactants (model acylating agent and -NH2 of amino acids) to initiate biologically relevant reactions during early evolution.

Hyperacetylation of the C-terminal domain of p53 inhibits the formation of the p53/p21 complex

Given our previous finding that certain tumor-suppressing functions of p53 are exerted by the p53/p21 complex, rather than p53 alone, cells may have a system to regulate the p53/p21 interaction. As p53 binds to p21 via its C-terminal domain, which contains acetylable lysine residues, we investigated whether the C-terminal acetylation of p53 influences the p53/p21 interaction. Indeed, the p53/p21 interaction was reduced when various types of cells (HCT116 colon cancer, A549 lung cancer, and MCF7 breast cancer cells) were treated with MS-275, an inhibitor of SIRT1 (a p53 deacetylase), or with SIRT1-targeting small interfering RNAs. These treatments also increased the acetylation levels of the five lysine residues (K370, K372, K373, K381, K382) in the C-terminal domain of p53. The p53/p21 interaction was also reduced when these lysine residues were substituted with glutamine (an acetylation memetic), but not arginine (an unacetylable lysine analog). While the inhibitory effect of the lysine-to-glutamine substitution was evident upon the substitution of all the five lysine residues, the substitution of only two (K381, K382) or three residues (K370, K372, K373) was less effective. Consistently, the five substitutions reduced the ability of p53 to regulate cell invasion and death by liberating Bax from Bcl-w. Overall, our data suggest that the acetylation, especially the hyperacetylation, of the p53 C-terminal domain suppresses the p53/p21-complex-dependent functions of p53 by inhibiting the p53/p21 interaction. We propose that cellular components involved in the acetylation or deacetylation of the p53 C-terminus are critical regulators of the formation of p53/p21 complex.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Solid-Phase Peptide Modification via Deaminative Photochemical Csp3 -Csp3 Bond Formation Using Katritzky Salts.

Introduction of unnatural amino acids can significantly improve the binding affinity and stability of peptides. Commercial availability of such amino acids is limited, and their synthesis is a long and tedious process. We here describe a method that allows the functionalization of peptides directly on solid‐support by converting lysine residues to Katritzky salts, and subjecting them to a photochemical Giese reaction under mild reaction conditions. The method avoids the need for amino acid synthesis and instead offers a late‐stage modification route for rapid peptide diversification. While numerous modification approaches at the lysine amine have been described, this work provides the first example of deaminative functionalization of peptides at lysine. The two‐step protocol is compatible with various substrates, lysine analogues, resins, and all proteinogenic amino acids. Finally, by leveraging solid‐phase modification, this protocol facilitates the functionalization of longer peptides as was demonstrated using biologically relevant peptides of up to 15 amino acids.

Structural basis of nucleosomal H4K20 methylation by methyltransferase SET8.

Histone H4lysine 20monomethylation (H4K20me1) plays a crucial role in multiple processes including DNA damage repair, DNA replication, and cell cycle control. Histone methyltransferase SET8 (previously named PR-Set7/KMT5A) mediates the chromatin deposition of H4K20me1, but how SET8 recognizes and modifies H4 in the context of the nucleosome is not fully understood. Here, we developed a simple chemical modification approach for H4K20substitution by using the lysine analog S-ethyl-L-cysteine (Ecx). Substitution of H4K20 with H4Ecx20 improves the stability of the SET8-nucleosome complex, allowing us to determine the cryo-EM structure at 3.2Å resolution. Structural analyses show that SET8 directly interacts with the H4 tail and the H2A-H2B acidic patch to ensure nucleosome binding. SET8 residues R339, K341, K351make contact with nucleosomal DNA at the super helical location 2 (SHL2). Substitution of SET8 DNA-binding residues with alanines decreases the SET8-nucleosome interaction and impairs the methyltransferase activity. Disrupting the binding between SET8 R192 and H2A-H2B acidic patch decreases the cellular level of H4K20me1. Together, these results reveal a near-atomic resolution structure of SET8-bound nucleosome and provide insights into the SET8-mediated H4K20 recognition and modification. The lysine-to-Ecx substitution approach can be applied to the study of other methyltransferases.