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Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late‐stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non‐native moieties. Utilising an allenamide, we report the first semisynthesis of antimicrobial lipopeptides leveraging a highly efficient thia‐Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide‐mediated thia‐Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide‐linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22 lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g. amines), ultimately yielding analogues with potent Gram‐negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a β‐keto amide on unprotected peptides, offering a powerful "jack‐of‐all‐trades" synthetic intermediate to enable further peptide modification. [ABSTRACT FROM AUTHOR]

Dysregulation of the CXCL12/CXCR4 axis is implicated in autoimmune, inflammatory, and oncogenic diseases, positioning CXCR4 as a pivotal therapeutic target. We evaluated optimized variants of the specific endogenous CXCR4 antagonist, EPI-X4, addressing existing challenges in stability and potency. Our structure-activity relationship study investigates the conjugation of EPI-X4 derivatives with long-chain fatty acids, enhancing serum albumin interaction and receptor affinity. Molecular dynamic simulations revealed that the lipid moieties stabilize the peptide-receptor interaction through hydrophobic contacts at the receptor's N-terminus, anchoring the lipopeptide within the CXCR4 binding pocket and maintaining essential receptor interactions. Accordingly, lipidation resulted in increased receptor affinities and antagonistic activities. Additionally, by interacting with human serum albumin lipidated EPI-X4 derivatives displayed sustained stability in human plasma and extended circulation times in vivo. Selected candidates showed significant therapeutic potential in human retinoblastoma cells in vitro and in ovo , with our lead derivative exhibiting higher efficacies compared to its non-lipidated counterpart. This study not only elucidates the optimization trajectory for EPI-X4 derivatives but also underscores the intricate interplay between stability and efficacy, crucial for delineating their translational potential in clinical applications. [Display omitted] • Lipidated derivatives of EPI-X4 exhibit increased affinity and antagonistic activity towards the CXCR4 receptor. • Lipidated EPI-X4 derivatives exhibit a half-life of >8 h in both blood and plasma. • The lipidated EPI-X4 JM#198 displays a circulation half-life of nearly 4 h in mice. • EPI-X4 JM#198 inhibits retinoblastoma tumor formation in vitro and in ovo. [ABSTRACT FROM AUTHOR]

Abstract Background Aging is a multifaceted process that affects all organ systems. With the increasing trend of population aging, aging-related diseases have resulted in significant medical challenges and socioeconomic burdens. Mesenchymal stromal cells (MSCs), due to their antioxidative stress, immunoregulatory, and tissue repair capabilities, hold promise as a potential anti-aging intervention. Methods In this study, we transplanted MSCs into naturally aged rats at 24 months, and subsequently examined levels of aging-related factors such as β-galactosidase, superoxide dismutase, p16, p21 and malondialdehyde in multiple organs. Additionally, we assessed various aging-related phenotypes in these aged rats, including immune senescence, lipid deposition, myocardial fibrosis, and tissue damage. We also conducted a 16 S ribosomal ribonucleic acid (rRNA) analysis to study the composition of gut microbiota. Results The results indicated that MSCs significantly reduced the levels of aging-associated and oxidative stress-related factors in multiple organs such as the heart, liver, and lungs of naturally aging rats. Furthermore, they mitigated chronic tissue damage and inflammation caused by aging, reduced levels of liver lipid deposition and myocardial fibrosis, alleviated aging-associated immunodeficiency and immune cell apoptosis, and positively influenced the gut microbiota composition towards a more youthful state. This research underscores the diverse anti-aging effects of MSCs, including oxidative stress reduction, tissue repair, metabolic regulation, and improvement of immune functions, shedding light on the underlying anti-aging mechanisms associated with MSCs. Conclusions The study confirms that MSCs hold great promise as a potential anti-aging approach, offering the possibility of extending lifespan and improving the quality of life in the elderly population.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host. [ABSTRACT FROM AUTHOR]

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

Macroautophagy/autophagy degrades and recycles cellular constituents via the lysosome to maintain cellular homeostasis. Our study identified the endoplasmic reticulum (ER)-resident SIGMAR1 (sigma non-opioid intracellular receptor 1) as a critical regulator of the biosynthesis of Atg8-family proteins that leads to the lipidation that is essential during autophagosome formation. We demonstrate that SIGMAR1 stabilizes MAP1LC3B/LC3B and GABARAP mRNAs, promoting their localized translation proximal to the ER for efficient lipidation. Using single-molecule fluorescence in situ hybridization/smFISH and co-immunoprecipitation, we found that SIGMAR1 directly binds to a conserved region in the 3’ UTR of LC3B mRNA, facilitating its translation, efficient lipidation, and proper integration into the phagophore membrane. Cells lacking SIGMAR1 show reduced levels of many Atg8-family proteins and impaired autophagic flux. Our model suggests that SIGMAR1-mediated localized translation of Atg8-family proteins at the ER promotes efficient autophagosome formation, in contrast to recruiting preexisting cytosolic Atg8-family proteins to the lipidation machinery. Elucidating the role of SIGMAR1 in autophagy may provide better therapeutic strategies to prevent or treat autophagy-dependent neurodegenerative diseases, particularly given the highly druggable nature of SIGMAR1. [ABSTRACT FROM AUTHOR]

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad‐spectrum and low resistance tendency. Herein, a library of fluoro‐lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non‐fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co‐assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA‐induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug‐resistant bacterial infections. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while nonlipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by pathophysiological deposits of extracellular amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles of tau. The central role of Aβ in AD pathology is well-established, with its increased deposition attributed mainly to its decreased cerebral clearance. Here, it is noteworthy that apolipoprotein E (ApoE), the most significant risk factor for AD, has been shown to play an isoform-specific role in clearing Aβ deposits (ApoE2 > ApoE3 > ApoE4), owing mainly to its lipidation status. In addition to the pathophysiological Aβ deposits, AD is also characterized by abnormal glucose metabolism, which is a distinct event preceding Aβ deposition. The present study established, for the first time, a possible link between these two major AD etiologies, with glucose metabolism directly influencing ApoE lipidation and its secretion by astrocytes expressing human ApoE4. Specifically, glucose dose-dependently activated liver X receptor (LXR), leading to elevated ABCA1 and ABCG1 protein levels and enhanced ApoE lipidation. Moreover, co-treatment with a glycolytic inhibitor significantly inhibited this LXR activation and subsequent ApoE lipidation, further supporting a central role of glucose metabolism in LXR activation leading to enhanced ApoE lipidation, which may help against AD through potential Aβ clearance. Therefore, we hypothesized that pharmacological agents that can target cellular energy metabolism, specifically aerobic glycolysis, may hold significant therapeutic potential against AD. In this context, the present study also led to the discovery of novel, small-molecule stimulants of astrocytic glucose metabolism, leading to significantly enhanced lipidation status of ApoE4 in astrocytic cells. Three such newly discovered compounds (lonidamine, phenformin, and berberine), owing to their promising cellular effect on the glycolysis-ApoE nexus, warrant further investigation in suitable in vivo models of AD. [ABSTRACT FROM AUTHOR]

Protein lipidation is a common co- or post-translational modification that plays a crucial role in regulating the localization, interaction and function of cellular proteins. Dysregulation of lipid modifications can lead to various diseases, including cancer, neurodegenerative diseases and infectious diseases. Therefore, the identification of proteins undergoing lipidation and their lipidation sites should provide insights into many aspects of lipid biology, as well as providing potential targets for therapeutic strategies. Bottom-up proteomics using liquid chromatography/tandem mass spectrometry is a powerful technique for the global analysis of protein lipidation. Here, we review proteomic methods for profiling protein lipidation, focusing on the two major approaches: the use of chemical probes, such as lipid alkyne probes, and the use of enrichment techniques for endogenous lipid-modified peptides. The challenges facing these methods and the prospects for developing them further to achieve a comprehensive analysis of lipid modifications are discussed. [ABSTRACT FROM AUTHOR]

Background: Apolipoprotein E4 (APOE4) is the most prevalent genetic risk factor of Alzheimer's disease. Several studies suggest that APOE4 binding to its receptors is associated with their internalization and accumulation in intracellular compartments. Importantly, this phenomenon also occurs with other, non-ApoE receptors. Based on these observations, we hypothesized that APOE4 pathological effects are mediated by impairment in the life cycle of distinct receptors (APOER2, LRP1, IR, VEGFR). Objective: To examine the effects of APOE genotype on receptors protein levels and compartmentalization. Methods: Primary mouse neurons were prepared from APOE3 or APOE4 targeted replacement mice, or APOE-KO mice. Specific receptors protein levels were evaluated in these neurons, utilizing immunofluorescent staining. Additionally, surface membrane protein levels of those receptors were assessed by cell surface biotinylation assay and ELISA. Receptors' colocalization with intracellular compartments was assessed by double staining and confocal microscopy, followed by colocalization analysis. Finally, LRP1 or APOER2 were knocked-down with CRISPR/Cas9 system to examine their role in mediating APOE4 effects on the receptors. Results: Our results revealed lower receptors' levels in APOE4, specifically on the membrane surface. Additionally, APOE4 affects the compartmentation of these receptors in two patterns: the first was observed with LRP1 and was associated with decreased receptor levels in numerous intracellular compartments. The second was obtained with the other receptors and was associated with their accumulation in early endosomes and their decrease in the late endosomes. Conclusions: These results provide a unifying mechanism, in which APOE4 drives the down regulation of various receptors, which plays important roles in distinct APOE4 related pathological processes. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while non-lipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

The demand for broad-spectrum antibacterial agents continues with increasing rates of resistance of microbial pathogens to traditional antibiotics. Peptides and lipopeptides are gaining traction as promising novel, class-reference antibiotics for tackling difficult-to-treat infections caused by multi-drug resistant bacteria. To identify novel candidates and expand treatment options in clinical settings, we explored the in vitro antibacterial potential and mode of action of a short octapeptide combining a cationic block of four lysines and a highly hydrophobic segment of four phenylalanines (K4F4), and two K4F4-inspired lipopeptides (Palmitoyl-K4F4 and K4-NH-Palmitoyl). Preliminary AI-based screening had revealed the antimicrobial potential of the K4F4 peptide coupled with limited haemolytic activity. Broth dilution and haemolytic assays have confirmed these in silico predictions. Overall, our lipidated peptides were more active at lower MIC values compared to non-lipidated species, indicating the beneficial impact of tailing lipidation on design of peptide-based antimicrobials. An integrated view of the membrane-active mechanism of these novel therapeutic templates was obtained using a combination of flow cytometry, fluorescence microscopy and dye-based permeabilization assays. K4F4 and its lipidated derivatives act via a fast-disrupting mechanism without inducing bacterial resistance mechanisms in a long-term exposure assay. A K4F4-inspired lipopeptide together with its shorter version (K4-NH-Palmitoyl), were more stable in environments closer emulating physiological conditions, showing a higher antibacterial response in physiological salts and serum than their parent peptide. Our findings reveal the antibacterial and antibiofilm potential of a novel polylysine-polyphenyalanine peptide and highlight the significant contribution of lipidation and shortening as molecular engineering strategies to improve and guide the future design of next-generation membrane-targeting antibiotics.

Caveolin‐1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin‐1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within the C‐terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full‐length S‐alkylated caveolin‐1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI‐LC‐MS analyses verified the addition of the C16 alkyl groups to caveolin‐1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed that S‐alkylation does not significantly affect the global helicity of the protein; however, molecular dynamics simulations revealed that there were local regions where the helicity was altered positively or negatively by S‐alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of the C‐terminal domain, resulting in a disposition within the bilayer characterized by increased depth. [ABSTRACT FROM AUTHOR]

Protein–lipid conjugation is a widespread modification involved in many biological processes. Various lipids, including fatty acids, isoprenoids, sterols, glycosylphosphatidylinositol, sphingolipids, and phospholipids, are covalently linked with proteins. These modifications direct proteins to intracellular membranes through the hydrophobic nature of lipids. Some of these membrane-binding processes are reversible through delipidation or by reducing the affinity to membranes. Many signaling molecules undergo lipid modification, and their membrane binding is important for proper signal transduction. The conjugation of proteins to lipids also influences the dynamics and function of organellar membranes. Dysregulation of lipidation has been associated with diseases such as neurodegenerative diseases. In this review, we first provide an overview of diverse forms of protein–lipid conjugation and then summarize the catalytic mechanisms, regulation, and roles of these modifications. Key words: lipid, lipidation, membrane, organelle, protein modification

Antimicrobial peptides (AMPs) are potentially powerful alternatives to conventional antibiotics in combating multidrug resistance, given their broad spectrum of activity. They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures, resulting in permeability and destruction of target microorganism membranes. Our earlier work showed that two leading AMPs, MSI-78 (4-20) and pardaxin (1-22), had potent antimicrobial activity against a range of bacteria. It is known that the attachment of moderate-length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer, inducing membrane curvature, destabilization, and potential leakage. Thus, in this work, we aimed to investigate the antimicrobial activity, oligomerization propensity, and lipidmembrane binding interactions of a range of N-terminal lipidated analogs of MSI-78 (4-20) and pardaxin (1-22). Molecular modeling results suggest that aggregation of the N-lipidated AMPs may impart greater structural stability to the peptides in solution and a greater depth of lipid bilayer insertion for the N-lipidated AMPs over the parental peptide. Our experimental and computational findings provide insights into how N-terminal lipidation of AMPs may alter their conformations, with subsequent effects on their functional properties in regard to their self-aggregation behavior, membrane interactions, and antimicrobial activity. [ABSTRACT FROM AUTHOR]

Microbiologically influenced corrosion is a common problem in the industrial field due to the deterioration of metals in the presence of various microorganisms, in particular sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB). A common method to reduce microbiologically influenced corrosion is the application of biocides. The limited number of suitable biocides and the resulting development of resistance, high dosage, and high application rate hinder an effective application. An environmentally friendly alternative could be the application of antimicrobial peptides (AMP), which have already been established in the field of medical devices for a while. Here, the successful treatment of different AMPs against 3 SRB and 1 SOB was demonstrated. The peptide L5K5W was favored due to its broad activity, high stability, and simple structure resulting in low synthesis costs. An alanine scan showed that substitution of leucine with tryptophan increased the activity of this peptide twofold compared to the original peptide against D. vulgaris, the main representative of SRB. Additional optimization of this modified peptide through changes in amino acid composition and lipidations significantly increased the effectiveness, finally resulting in a minimum inhibitory concentration (MIC) of 15.63 μg/mL against Desulfovibrio vulgaris. Even against the marine SRB Desulfovibrio indonesiensis with a required salt concentration of min. 2%, an activity of the peptides can be observed (MIC: 31.25 μg/mL). The peptides also remained stable and active for 7 days in the supernatant of the bacterial culture. Key points: • Antimicrobial peptides provide an alternative to combat biocorrosive bacteria. • Optimization of the peptide sequence leads to a significant increase in activity. • The investigated peptides exhibit high stability, both in the medium and in the bacterial supernatant. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) have recently gained attention as a viable solution for combatting antibiotic resistance due to their numerous advantages, including their broad-spectrum activity, low propensity for inducing resistance, and low cytotoxicity. Unfortunately, their clinical application is limited due to their short half-life and susceptibility to proteolytic cleavage by serum proteases. Indeed, several chemical strategies, such as peptide cyclization, N-methylation, PEGylation, glycosylation, and lipidation, are widely used for overcoming these issues. This review describes how lipidation and glycosylation are commonly used to increase AMPs' efficacy and engineer novel AMP-based delivery systems. The glycosylation of AMPs, which involves the conjugation of sugar moieties such as glucose and N-acetyl galactosamine, modulates their pharmacokinetic and pharmacodynamic properties, improves their antimicrobial activity, and reduces their interaction with mammalian cells, thereby increasing selectivity toward bacterial membranes. In the same way, lipidation of AMPs, which involves the covalent addition of fatty acids, has a significant impact on their therapeutic index by influencing their physicochemical properties and interaction with bacterial and mammalian membranes. This review highlights the possibility of using glycosylation and lipidation strategies to increase the efficacy and activity of conventional AMPs. [ABSTRACT FROM AUTHOR]

Abstract Antimicrobial peptides (AMPs) are potentially powerful alternatives to conventional antibiotics in combating multidrug resistance, given their broad spectrum of activity. They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures, resulting in permeability and destruction of target microorganism membranes. Our earlier work showed that two leading AMPs, MSI‐78 (4–20) and pardaxin (1–22), had potent antimicrobial activity against a range of bacteria. It is known that the attachment of moderate‐length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer, inducing membrane curvature, destabilization, and potential leakage. Thus, in this work, we aimed to investigate the antimicrobial activity, oligomerization propensity, and lipid‐membrane binding interactions of a range of N‐terminal lipidated analogs of MSI‐78 (4–20) and pardaxin (1–22). Molecular modeling results suggest that aggregation of the N‐lipidated AMPs may impart greater structural stability to the peptides in solution and a greater depth of lipid bilayer insertion for the N‐lipidated AMPs over the parental peptide. Our experimental and computational findings provide insights into how N‐terminal lipidation of AMPs may alter their conformations, with subsequent effects on their functional properties in regard to their self‐aggregation behavior, membrane interactions, and antimicrobial activity.

An effective vaccine against group A streptococcus (GAS) is highly desirable for definitive control of GAS infections. In the present study, two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed. The vaccines were primarily composed of encapsulated KLH protein (a source of T helper cell epitopes) and lipidated M-protein derived B cell peptide epitope (lipoJ14) within the amphiphilic structure of nanoparticles. The only difference between them was one of the nanoparticles vaccines received additional surface coating with poly (I:C). The formulated vaccines exhibited nanosized particles within the range of 220–240 nm. Cellular uptake study showed that nanoparticles vaccine without additional poly (I:C) coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly (I:C). Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells. Upon immunization in mice, both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture. These antibodies elicited high opsonic activity against the tested GAS strains. Overall, our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly (I:C).

The protein cell division cycle 42 (Cdc42) is a small GTPase of the Rho family regulating a plethora of physiological functions in a tissue, cell and subcellular-specific manner via participating in multiple signaling pathways. Since the corresponding signaling hubs are mainly organized along the cellular membranes, cytosolic proteins like Cdc42 need to be properly targeted and held at the membrane. Here, lipid modifications come into play: Cdc42 can be associated with membranes by different lipid anchors including prenylation (Cdc42-prenyl) and palmitoylation (Cdc42-palm). While Cdc42-prenyl is ubiquitously expressed, Cdc42-palm splicing variant in mainly expressed in the brain. Mechanisms underlying Cdc42 lipidation as well as its regulation are the main topic of this review. Furthermore, we will discuss the functional importance of Cdc42 lipid modifications with the focus on the role of different lipids in regulating defined Cdc42 functions. Finally, we will provide an overview of the possible implementation of Cdc42 lipidation in pathological conditions and different diseases.

During autophagy, the ATG8 family proteins have several well-characterized roles in facilitating early, mid, and late steps of autophagy, including autophagosome expansion, cargo recruitment and autophagosome-lysosome fusion. Their discovery has importantly allowed for precise experimental monitoring of the pathway, bringing about a huge expansion of research in the field over the last decades. In this review, we discuss both canonical and non-canonical roles of the autophagic lipidation machinery, with particular focus on the ATG8 proteins, their post-translational modifications and their increasingly uncovered alternative roles mediated through their anchoring at different membranes. These include endosomes, macropinosomes, phagosomes and the plasma membrane, to which ATG8 proteins can bind through canonical or alternative lipidation. Beyond new ATG8 binding partners and cargo types, we also explore several open questions related to alternative outcomes of autophagic machinery engagement beyond degradation. These include their roles in plasma membrane repair and secretion of selected substrates as well as the physiological implications hereof in health and disease.

Optimizing the druggability of both native and AI-designed bioactive proteins is crucial for realizing their therapeutic potential. A key focus in designing protein-based therapeutics is improving their pharmacokinetic properties. However, a significant challenge is to preserve biological activity while implementing long-acting strategies. Fibroblast growth factor 21 (FGF21), an endogenous hormone with potential as a treatment for non-alcoholic steatohepatitis (NASH), exemplifies this challenge. In this study, we present a novel lipidation-dimerization (LiDi) platform that integrates lipidation with a dimeric form of FGF21 connected by a hydrophilic linker. The lipidation enhances albumin binding, enabling sustained release, while the dimeric structure boosts biological activity. In vivo evaluations of the LiDi FGF21 analogs demonstrated that they offer excellent pharmacokinetic properties and superior efficacy compared to other treatments for NASH. This platform effectively extends the therapeutic half-life of proteins without compromising their activity, substantially broadening the application range of proteins as therapeutics., Competing Interests: Declaration of competing interest Fei Zhao and Yunliang Qiu own shares of Shanghai InnoStar Bio-tech Co., Ltd. Jianguang Lu owns shares of Shanghai Duomirui Bio-tech Co., Ltd., (Copyright © 2024. Published by Elsevier B.V.)

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi., Competing Interests: DW, AP No competing interests declared, (© 2024, Williams and Peden.)

Human serum albumin (hSA) is the major carrier protein for fatty acids (FAs) in plasma. Its ability to bind multiple FA moieties with moderate to high affinity has inspired the use of FA conjugation as a safe and natural platform to generate long-lasting therapeutics with enhanced pharmacokinetic properties and superior efficacy. In this frame, the choice of the FA is crucial and a comprehensive elucidation of the molecular interactions of FAs with hSA cannot be left out of consideration. To this intent, we report here a comparative analysis of the binding mode of different FA moieties with hSA. The choice among different albumin-binding FAs and how this influence the pharmacokinetics properties of a broad spectrum of therapeutic molecules will be discussed including a critical description of some clinically relevant FA conjugated therapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3′ UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3′ UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3′ UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3′ UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease. [Display omitted] • SigmaR1 (S1R) is an RNA-binding ER membrane protein that recruits LC3B mRNA to the ER • S1R interacts with the 3′ UTR of LC3B mRNA to promote its translation • Without S1R, translation of LC3B is repressed in vitro and in cells • The 3′ UTR of LC3B mRNA is required for proper lipidation of the LC3B protein product Knupp and Chen et al. have uncovered a role for the ER membrane protein SigmaR1 (S1R) in promoting the localized translation of Atg8 family proteins. S1R directly binds to the 3′ UTR of LC3B mRNA to promote ER-localized translation of the LC3B protein product. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) have recently gained attention as a viable solution for combatting antibiotic resistance due to their numerous advantages, including their broad-spectrum activity, low propensity for inducing resistance, and low cytotoxicity. Unfortunately, their clinical application is limited due to their short half-life and susceptibility to proteolytic cleavage by serum proteases. Indeed, several chemical strategies, such as peptide cyclization, N-methylation, PEGylation, glycosylation, and lipidation, are widely used for overcoming these issues. This review describes how lipidation and glycosylation are commonly used to increase AMPs’ efficacy and engineer novel AMP-based delivery systems. The glycosylation of AMPs, which involves the conjugation of sugar moieties such as glucose and N-acetyl galactosamine, modulates their pharmacokinetic and pharmacodynamic properties, improves their antimicrobial activity, and reduces their interaction with mammalian cells, thereby increasing selectivity toward bacterial membranes. In the same way, lipidation of AMPs, which involves the covalent addition of fatty acids, has a significant impact on their therapeutic index by influencing their physicochemical properties and interaction with bacterial and mammalian membranes. This review highlights the possibility of using glycosylation and lipidation strategies to increase the efficacy and activity of conventional AMPs.

An effective vaccine against group A streptococcus (GAS) is highly desirable for definitive control of GAS infections. In the present study, two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed. The vaccines were primarily composed of encapsulated KLH protein (a source of Thelper cell epitopes) and lipidated M-protein derived B cell peptide epitope (lipoJ14) within the amphiphilic structure of nanoparticles. The only difference between them was one of the nanoparticles vaccines received additional surface coating with poly (I:C). The formulated vaccines exhibited nanosized particles within the range of 220-240 nm. Cellular uptake study showed that nanoparticles vaccine without additional poly (I:C) coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly (I:C). Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells. Upon immunization in mice, both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture. These antibodies elicited high opsonic activity against the tested GAS strains. Overall, our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly (I:C). [ABSTRACT FROM AUTHOR]

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N -acetylglucosamine or N -acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS. [ABSTRACT FROM AUTHOR]

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action. [ABSTRACT FROM AUTHOR]

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

The lipid post‐translational modification S‐palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review, we discuss the past and recent advances in the experimental tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy. [ABSTRACT FROM AUTHOR]

Protein S -acylation is a prevalent post-translational protein lipidation that is dynamically regulated by 'writer' protein S -acyltransferases and 'eraser' acylprotein thioesterases. The protein S -acyltransferases comprise 23 aspartate–histidine–histidine–cysteine (DHHC)–containing proteins, which transfer fatty acid acyl groups from acyl-coenzyme A onto protein substrates. DHHC proteins are increasingly recognized as critical regulators of S -acylation–mediated cellular processes and pathology. As our understanding of the importance and breadth of DHHC-mediated biology and pathology expands, so too does the need for chemical inhibitors of this class of proteins. In this review, we discuss the challenges and progress in DHHC inhibitor development, focusing on 2-bromopalmitate, the most commonly used inhibitor in the field, and N -cyanomethyl- N -myracrylamide, a new broad-spectrum DHHC inhibitor. We believe that current and ongoing advances in structure elucidation, mechanistic interrogation, and novel inhibitor design around DHHC proteins will spark innovative strategies to modulate these critical proteins in living systems. [ABSTRACT FROM AUTHOR]

Liver-derived serum amyloid A (SAA) is present in plasma where it is mainly associated with HDL and from which it is cleared more rapidly than are the other major HDL-associated apolipoproteins. Although evidence suggests that lipid-free and HDL-associated forms of SAA have different activities, the pathways by which SAA associates and disassociates with HDL are poorly understood. In this study, we investigated SAA lipidation by hepatocytes and how this lipidation relates to the formation of nascent HDL particles. We also examined hepatocyte-mediated clearance of lipid-free and HDL-associated SAA. We prepared hepatocytes from mice injected with lipopolysaccharide or an SAA-expressing adenoviral vector. Alternatively, we incubated primary hepatocytes from SAA-deficient mice with purified SAA. We analyzed conditioned media to determine the lipidation status of endogenously produced and exogenously added SAA. Examining the migration of lipidated species, we found that SAA is lipidated and forms nascent particles that are distinct from apoA-I-containing particles and that apoA-I lipidation is unaltered when SAA is overexpressed or added to the cells, indicating that SAA is not incorporated into apoA-I-containing HDL during HDL biogenesis. Like apoA-I formation, generation of SAA-containing particles was dependent on ABCA1, but not on scavenger receptor class B type I. Hepatocytes degraded significantly more SAA than apoA-I. Taken together, our results indicate that SAA's lipidation and metabolism by the liver is independent of apoA-I and that SAA is not incorporated into HDL during HDL biogenesis.

Molecular dynamic behaviors of nanodisc (ND) formulations of free doxorubicin (DOX) and DOX conjugated lipid prodrug molecules were investigated by molecular dynamics (MD) simulations. We have unveiled how formulation design affects the drug release profile and conformational stability of ND assemblies. Our simulation results indicate that free DOX molecules loaded in the ND system experienced rapid dissociation due to the unfavorable orientation of DOX attached to the lipid surface. It is found that DOX tends to form aggregates with higher drug quantities. In contrast, lipidated DOX-prodrugs incorporated in ND formulations exhibited sufficient ND conformational stability. The drug loading capacity is dependent on the type of lipid molecules grafted on the DOX-prodrug, and the drug loading quantities in a fixed area of NDs follow the order: DOX-BMPH-MP > DOX-BMPH-TC > DOX-BMPH-PTE. To gain further insight into the dynamic characteristics of ND formulations governed by different kinds of lipidation, we investigated the conformational variation of ND components, intermolecular interactions, the solvent accessible surface area, and individual MSP1 residue flexibility. We found that the global conformational stability of DOX-prodrug-loaded ND assemblies is influenced by the molecular flexibility and lipidated forms of DOX-prodrug. We also found that the spontaneous self-aggregation of DOX-prodrugs with increasing quantities on ND could reduce the membrane fluidity and enhance the conformational stability of ND formulations.

Abstract The ER is hub for protein folding. Proteins that harbor a Frizzled cysteine-rich domain (FZ-CRD) possess 10 conserved cysteine motifs held by a unique disulfide bridge pattern which attains a correct fold in the ER. Little is known about implications of disease-causing missense mutations within FZ-CRD families. Mutations in FZ-CRD of Frizzled class receptor 4 (FZD4) and Muscle, skeletal, receptor tyrosine kinase (MuSK) and Receptor tyrosine kinase-like orphan receptor 2 (ROR2) cause Familial Exudative Vitreoretinopathy (FEVR), Congenital Myasthenic Syndrome (CMS), and Robinow Syndrome (RS) respectively. We highlight reported pathogenic inherited missense mutations in FZ-CRD of FZD4, MuSK and ROR2 which misfold, and traffic abnormally in the ER, with ER-associated degradation (ERAD) as a common pathogenic mechanism for disease. Our review shows that all studied FZ-CRD mutants of RS, FEVR and CMS result in misfolded proteins and/or partially misfolded proteins with an ERAD fate, thus we coin them as “disorders of FZ-CRD”. Abnormal trafficking was demonstrated in 17 of 29 mutants studied; 16 mutants were within and/or surrounding the FZ-CRD with two mutants distant from FZ-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. FZD4 and MuSK mutants were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. Investigating the cellular and molecular mechanisms of these mutations is important since misfolded protein and ER-targeted therapies are in development. The P344R-MuSK kinase mutant showed around 50% of its in-vitro autophosphorylation activity and P344R-MuSK increased two-fold on proteasome inhibition. M105T-FZD4, C204Y-FZD4, and P344R-MuSK mutants are thermosensitive and therefore, might benefit from extending the investigation to a larger number of chemical chaperones and/or proteasome inhibitors. Nonetheless, FZ-CRD ER-lipidation it less characterized in the literature and recent structural data sheds light on the importance of lipidation in protein glycosylation, proper folding, and ER trafficking. Current treatment strategies in-place for the conformational disease landscape is highlighted. From this review, we envision that disorders of FZ-CRD might be receptive to therapies that target FZ-CRD misfolding, regulation of fatty acids, and/or ER therapies; thus paving the way for a newly explored paradigm to treat different diseases with common defects.

Peptides, despite their therapeutic potential, face challenges with undesirable pharmacokinetic (PK) properties and biodistribution, including poor oral absorption and cellular uptake, and short plasma elimination half-lives. Lipidation of peptides is a common strategy to improve their physicochemical and PK properties, making them viable drug candidates. For example, the plasma half-life of peptides has been extended via conjugation to lipids that are proposed to promote binding to serum albumin and thus protect against rapid clearance. Recent work has shown that lipid conjugation to oligodeoxynucleotides, polymers and small molecule drugs results in association not only with albumin, but also with lipoproteins, resulting in half-life prolongation and transport from administration sites via the lymphatics. Enhancing delivery into the lymph increases the efficacy of vaccines and therapeutics with lymphatic targets such as immunotherapies. In this study, the plasma PK, lymphatic uptake, and bioavailability of the glucagon-like peptide-1 (GLP-1) receptor agonist peptides, liraglutide (lipidated) and exenatide (non-lipidated), were investigated following subcutaneous (SC) administration to rats. As expected, liraglutide displayed an apparent prolonged plasma half-life (9.1 versus 1 h), delayed peak plasma concentrations and lower bioavailability (∼10 % versus ∼100 %) compared to exenatide after SC administration. The lymphatic uptake of both peptides was relatively low (<0.5 % of the dose) although lymph to plasma concentration ratios were greater than one for several early timepoints suggesting some direct uptake into lymph. The low lymphatic uptake may be due to the nature of the conjugated lipid (a single-chain C16 palmitic acid in liraglutide) but suggests that other peptides with similar lipid conjugations may also have relatively modest lymphatic uptake. If delivery to the lymph is desired, conjugation to more lipophilic moieties with higher albumin and/or lipoprotein binding efficiencies, such as diacylglycerols, may be appropriate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Most of the functions of LC3/GABARAP in macroautophagy/autophagy are considered to depend on their association with the phagophore membrane through a conjugation to a lipid. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1, the single homolog of GABARAP in C. elegans. Mutants that express only cytosolic forms revealed an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. [ABSTRACT FROM AUTHOR]

The interior of the cell abounds with reactive species that can accumulate as non-enzymatic covalent modifications (NECMs) on biological macromolecules. These adducts interfere with many cellular processes, for example, by altering proteins' surface topology, enzymatic activity, or interactomes. Here, we discuss dynamic NECMs on chromatin, which serves as the cellular blueprint. We first outline the chemistry of NECM formation and then focus on the recently identified effects of their accumulation on chromatin structure and transcriptional output. We next describe the known cellular regulatory mechanisms that prevent or reverse NECM formation. Finally, we discuss recently developed chemical biology platforms for probing and manipulating these NECMs in vitro and in vivo. Post-translational modifications (PTMs) of proteins are introduced either by the specific activity of 'writer' enzymes or spontaneous chemical reaction with reactive small molecules forming non-enzymatic covalent modifications (NECMs). Histones are particularly susceptible to accumulate NECMs due to their long half-lives, unstructured tails, and abundance of accessible reactive arginine and lysine residues that are key sites for regulatory enzymatic PTMs. NECMs on histones compete with enzymatic PTMs as well as alter chromatin structure, subsequently affecting tightly regulated genetic processes and ultimately resulting in global cellular phenotype changes. NECMs can be enzymatically 'erased' as well as prevented by scavenger systems, including both small molecules and proteins. Advances in biophysical methods, immunoassays, mass spectrometry, chemical probe development, and chemoproteomics have established a platform that can be leveraged to predict, track, manipulate, and study histone NECMs. [ABSTRACT FROM AUTHOR]

Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.