Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications.

Lysine dimethylation (Kme2) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2, facilitating the global identification of low-abundance Kme2-PTMs and their sites within complex cell lysate mixtures.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

3-Acyl-4-Pyranone as a Lysine Residue-Selective Bioconjugation Reagent for Peptide and Protein Modification.

Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide-alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.

Chemical synthesis of site-selective advanced glycation end products in α-synuclein and its fragments.

Advanced glycation end products (AGEs) arise from the Maillard reaction between dicarbonyls and proteins, nucleic acids, or specific lipids. Notably, AGEs are linked to aging and implicated in various disorders, spanning from cancer to neurodegenerative diseases. While dicarbonyls like methylglyoxal preferentially target arginine residues, lysine-derived AGEs, such as N(6)-(1-carboxymethyl)lysine (CML) and N(6)-(1-carboxyethyl)lysine (CEL), are also abundant. Predicting protein glycation in vivo proves challenging due to the intricate nature of glycation reactions. In vitro, glycation is difficult to control, especially in proteins that harbor multiple glycation-prone amino acids. α-Synuclein (aSyn), pivotal in Parkinson's disease and synucleinopathies, has 15 lysine residues and is known to become glycated at multiple lysine sites. To understand the influence of glycation in specific regions of aSyn on its behavior, a strategy for site-specific glycated protein production is imperative. To fulfill this demand, we devised a synthetic route integrating solid-phase peptide synthesis, orthogonal protection of amino acid side-chain functionalities, and reductive amination strategies. This methodology yielded two disease-related N-terminal peptide fragments, each featuring five and six CML and CEL modifications, alongside a full-length aSyn protein containing a site-selective E46CEL modification. Our synthetic approach facilitates the broad introduction of glycation motifs at specific sites, providing a foundation for generating glycated forms of synucleinopathy-related and other disease-relevant proteins.

Tunable structure and linear viscoelastic properties of poly(glycerol adipate urethane)-based elastomeric composites for tissue regeneration.

Elastomeric biocomposites based on poly(glycerol adipate urethane) and hydroxyapatite were fabricated for tissue regeneration. The poly(glycerol adipate urethane) (PGAU) elastomeric composite matrices were obtained by chemical crosslinking of the poly(glycerol adipate) prepolymer (pPGA) with diisocyanate derivative of L-lysine. Two series of composites varying in the amount of L-lysine diisocyanate ethyl ester (LDI) used as a crosslinking agent were manufactured. As a ceramic filler both unmodified and L-lysine surface-modified hydroxyapatite (HAP) particles were used. The novelty of our research consists in the manufactured elastomeric materials and characterization of their linear viscoelastic (LVE) properties. The LVE properties of the composites were investigated by means of dynamic thermomechanical analysis. Frequency sweep and amplitude sweep measurements were performed in shear mode. The influence of the crosslinking agent (LDI) amount, HAP content and surface modification of HAP on the LVE properties of the composites was determined based on the analysis of the master curves of storage (G') and loss (G″) moduli and of tanδ of the composites. Depending on the amount of LDI, HAP and surface modification, the materials differ in the values of rubber elasticity plateau modulus (G0) and G' and G″ determined at selected shear frequencies and at the glassy state. G0 ranges from 278 kPa to 3.98 MPa, G' in the glassy state is within the range of 219 MPa-459 MPa. The G0 values of the PGAU-based composites are within the stiffness range of soft tissue. In view of the choice of HAP as the ceramic component and the G0 values, elastomeric composites have the potential to be used as filling materials in small bone defects (due to their mechanical similarity to osteoid) as well as materials for cartilage tissue regeneration.

Regulation of urea cycle by reversible high-stoichiometry lysine succinylation.

The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.

Quantifying carboxymethyl lysine and carboxyethyl lysine in human plasma: clinical insights into aging research using liquid chromatography-tandem mass spectrometry.

OBJECTIVE: The objective of this study was to establish a methodology for determining carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) concentrations in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test results were also used for clinical aging research. METHODS: Human plasma samples were incubated with aqueous perfluorovaleric acid (NFPA), succeeded by precipitation utilizing trichloroacetic acid, hydrolysis facilitated by hydrochloric acid, nitrogen drying, and ultimate re-dissolution utilizing NFPA, followed by filtration. Cotinine-D3 was added as an internal standard. The separation was performed on an Agela Venusil ASB C18 column (50 mm × 4.6 mm, 5 µm) with a 5 mmol/L NFPA and acetonitrile/water of 60:40 (v/v) containing 0.15% formic acid. The multiple reaction monitoring mode was used for detecting CML, CEL, and cotinine-D3, with ion pairs m/z 205.2 > 84.1 (for quantitative) and m/z 205.2 > m/z 130.0 for CML, m/z 219.1 > 84.1 (for quantitative) and m/z 219.1 > m/z 130.1 for CEL, and m/z 180.1 > 80.1 for cotinine-D3, respectively. RESULTS: The separation of CML and CEL was accomplished within a total analysis time of 6 minutes. The retention times of CML, CEL, and cotinine-D3 were 3.43 minutes, 3.46 minutes, and 4.50 minutes, respectively. The assay exhibited linearity in the concentration range of 0.025-1.500 µmol/L, with a lower limit of quantification of 0.025 µmol/L for both compounds. The relative standard deviations of intra-day and inter-day were both below 9%, and the relative errors were both within the range of ±4%. The average recoveries were 94.24% for CML and 97.89% for CEL. CONCLUSION: The results indicate that the developed methodology is fast, highly sensitive, highly specific, reproducible, and suitable for the rapid detection of CML and CEL in clinical human plasma samples. The outcomes of the clinical research project on aging underscored the important indicative significance of these two indicators for research on human aging.

Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action.

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization.

Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC-MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

Systematic enhancement of protein crystallization efficiency by bulk lysine-to-arginine (KR) substitution.

Structural genomics consortia established that protein crystallization is the primary obstacle to structure determination using x-ray crystallography. We previously demonstrated that crystallization propensity is systematically related to primary sequence, and we subsequently performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. This software can be run interactively on the worldwide web at https://www.pxengineering.org/. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization, and, for two of these three proteins, the construct with the largest number of KR substitutions exhibits significantly enhanced crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from Bulk KR-substituted domains show the engineered arginine residues frequently make hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that Bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve crystallization.

l-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation.

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

Submillisecond Atomistic Molecular Dynamics Simulations Reveal Hydrogen Bond-Driven Diffusion of a Guest Peptide in Protein-RNA Condensate.

Liquid-liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed accumulated 564.7 µs all-atom molecular dynamics (MD) simulations (system size ∼200k atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds and bind the strongest to the scaffold RNA in the condensate, relative to the Lys- and ADMA-rich peptides. Our MD simulations also showed that the Arg-rich peptide diffused more slowly in the condensate relative to the other two guest peptides, which is consistent with a recent fluorescence microscopy study. There was no significant increase in the number of cation-π interactions between the Arg-rich peptide and the scaffold RNA compared to the Lys-rich and ADMA-rich peptides. Our results indicate that hydrogen bonds between the peptides and the RNA backbone, rather than cation-π interactions, play a major role in regulating peptide diffusion in the condensate.

Peptide Stapling by Crosslinking Two Amines with α-Ketoaldehydes through Diverse Modified Glyoxal-Lysine Dimer Linkers.

α-Ketoaldehydes play versatile roles in the ubiquitous natural processes of protein glycation. However, leveraging the reactivity of α-ketoaldehydes for biomedical applications has been challenging. Previously, the reactivity of α-ketoaldehydes with guanidine has been harnessed to design probes for labeling Arg residues on proteins in an aqueous medium. Herein, a highly effective, broadly applicable, and operationally simple protocol for stapling native peptides by crosslinking two amino groups through diverse imidazolium linkers with various α-ketoaldehyde reagents is described. The use of hexafluoroisopropanol as a solvent facilitates rapid and clean reactions under mild conditions and enables unique selectivity for Lys over Arg. The naturally occurring GOLD/MOLD linkers have been expanded to encompass a wide range of modified glyoxal-lysine dimer (OLD) linkers. In a proof-of-concept trial, these modular stapling reactions enabled a convenient two-round strategy to streamline the structure-activity relationship (SAR) study of the wasp venom peptide anoplin, leading to enhanced biological activities.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids.

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome.

Synthesis of Amino Acid-Based Cationic Lipids and Study of the Role of the Cationic Head Group for Enhanced Drug and Nucleic Acid Delivery.

Leveraging liposomes for drug and nucleic acid delivery, though promising due to reduced toxicity and ease of preparation, faces challenges in stability and efficiency. To address this, we synthesized cationic amphiphiles from amino acids (arginine, lysine, and histidine). Histidine emerged as the superior candidate, leading to the development of three histidine-rich cationic amphiphiles for liposomes. Using the hydration method, we have prepared the liposomes and determined the optimal N/P ratios for lipoplex formation via gel electrophoresis. In vitro transfection assays compared the efficacy of our lipids to Fugene, while MTT assays gauged biocompatibility across cancer cell lines (MDA-MB 231 and MCF-7). The histidine-based lipid demonstrated marked potential in enhancing drug and nucleic acid delivery. This improvement stemmed from increased zeta potential, enhancing electrostatic interactions with nucleic acids and cellular uptake. Our findings underscore histidine's crucial role over lysine and arginine for effective delivery, revealing a significant correlation between histidine abundance and optimal performance. This study paves the way for histidine-enriched lipids as promising candidates for efficient drug and nucleic acid delivery, addressing key challenges in the field.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

Specific Inhibitors of Mitochondrial Deacylase Sirtuin 4 Endowed with Cellular Activity.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in aging-related diseases. Mammalian Sirtuin 4 (Sirt4) is located in mitochondria and a potential therapeutic target for cancer and metabolic diseases, but no potent and selective Sirt4 inhibitors have been reported. Here, we describe the identification of potent Sirt4-specific small-molecule inhibitors. Testing hits from a target-based virtual screen revealed 12 active compounds. A focused screen based on two top compounds, followed by structure-assisted design of derivatives, yielded four first-in-class potent Sirt4 inhibitors. Kinetic analyses indicate compound competition with the acyl peptide substrate, consistent with the docking models and implicating Sirt4's unique acyl binding site. The compounds indeed show preference for Sirt4 over other isoforms, with one of them (69) being highly isoform selective, and they are active in cells. Our results provide first lead compounds and mechanistic insights for optimization toward Sirt4-specific inhibitors useful as experimental tools and potential therapeutics.

A Structure-Activity Relationship Study of SNAIL1 Peptides as Inhibitors of Lysine-Specific Demethylase 1.

Peptides have recently garnered attention as middle-molecular-weight drugs with the characteristics of small molecules and macromolecules. Lysine-specific demethylase 1 (LSD1) is a potential therapeutic target for lung cancer, neuroblastoma, and leukemia, and some peptide-based LSD1 inhibitors designed based on the N-terminus of SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors, have been reported. The N-terminus of SNAIL1 peptide acts as a cap of the catalytic site of LSD1, inhibiting interactions with LSD1. However, the structure-activity relationship (SAR) of these inhibitors is not yet fully understood. Therefore, in the present study, we aimed to uncover the SAR and to identify novel SNAIL1 peptide-based LSD1 inhibitors. We synthesized peptide inhibitor candidates based on truncating the N-terminus of SNAIL1 or substituting its amino acid residues. In the truncation study, we found that SNAIL1 1-16 (2), which was composed of 16 residues, strongly inhibited LSD1. Furthermore, we investigated the SAR at residues-3 and -5 from the N-terminus and found that peptides 2j and 2k, in which leucine 5 of the parent peptide is substituted with unnatural amino acids, cyclohexylalanine and norleucine, respectively, strongly inhibited LSD1. This result suggests that the hydrophobic interaction between the inhibitor peptides and LSD1 affects the LSD1-inhibitory activity. We believe that this SAR information provides a basis for the development of more potent LSD1 inhibitors.

Analysis and review of techniques and tools based on machine learning and deep learning for prediction of lysine malonylation sites in protein sequences.

The post-translational modifications occur as crucial molecular regulatory mechanisms utilized to regulate diverse cellular processes. Malonylation of proteins, a reversible post-translational modification of lysine/k residues, is linked to a variety of biological functions, such as cellular regulation and pathogenesis. This modification plays a crucial role in metabolic pathways, mitochondrial functions, fatty acid oxidation and other life processes. However, accurately identifying malonylation sites is crucial to understand the molecular mechanism of malonylation, and the experimental identification can be a challenging and costly task. Recently, approaches based on machine learning (ML) have been suggested to address this issue. It has been demonstrated that these procedures improve accuracy while lowering costs and time constraints. However, these approaches also have specific shortcomings, including inappropriate feature extraction out of protein sequences, high-dimensional features and inefficient underlying classifiers. As a result, there is an urgent need for effective predictors and calculation methods. In this study, we provide a comprehensive analysis and review of existing prediction models, tools and benchmark datasets for predicting malonylation sites in protein sequences followed by a comparison study. The review consists of the specifications of benchmark datasets, explanation of features and encoding methods, descriptions of the predictions approaches and their embedding ML or deep learning models and the description and comparison of the existing tools in this domain. To evaluate and compare the prediction capability of the tools, a new bunch of data has been extracted based on the most updated database and the tools have been assessed based on the extracted data. Finally, a hybrid architecture consisting of several classifiers including classical ML models and a deep learning model has been proposed to ensemble the prediction results. This approach demonstrates the better performance in comparison with all prediction tools included in this study (the source codes of the models presented in this manuscript are available in https://github.com/Malonylation). Database URL: https://github.com/A-Golshan/Malonylation.

Internal Fragments Enhance Middle-Down Mass Spectrometry Structural Characterization of Monoclonal Antibodies and Antibody-Drug Conjugates.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (∼150 kDa) and high structural complexity that require extensive sequence and structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique that sequences and maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications that may occur in bottom-up MS while achieving higher sequence coverage compared to top-down MS. However, returning complete sequence information by MD-MS is still challenging. Here, we show that assigning internal fragments in direct infusion MD-MS of a mAb and an ADC substantially improves their structural characterization. For MD-MS of the reduced NIST mAb, including internal fragments recovers nearly 100% of the sequence by accessing the middle sequence region that is inaccessible by terminal fragments. The identification of important glycosylations can also be improved after the inclusion of internal fragments. For the reduced lysine-linked IgG1-DM1 ADC, we show that considering internal fragments increases the DM1 conjugation sites coverage to 80%, comparable to the reported 83% coverage achieved by peptide mapping on the same ADC (Luo et al. Anal. Chem. 2016, 88, 695-702). This study expands our work on the application of internal fragment assignments in top-down MS of mAbs and ADCs and can be extended to other heterogeneous therapeutic molecules such as multispecifics and fusion proteins for more widespread applications.