N-terminal Residues Research Articles

Photochemical and Collision-Induced Cross-Linking of Lys, Arg, and His to Nitrile Imines in Peptide Conjugate Ions in the Gas Phase.

We report a study of internal covalent cross-linking with photolytically generated diarylnitrile imines of N-terminal arginine, lysine, and histidine residues in peptide conjugates. Conjugates in which a 4-(2-phenyltetrazol-5-yl)benzoyl group was attached to C-terminal lysine, that we call RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K, were ionized by electrospray and subjected to UV photodissociation (UVPD) at 213 nm. UVPD triggered loss of N2 and proceeded by covalent cross-linking to nitrile imine intermediates that involved the side chains of N-terminal arginine, lysine, and histidine, as well as the peptide amide groups. Cross-linking yields were determined from UVPD-MS2 measurements as 67%, 66%, and 84% for RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K ions, respectively. CID-MS3 of the denitrogenated ion intermediates from RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K indicated overall cross-linking yields of 80%, 89%, and 80%, respectively. The nature of the cross-linking reactions and cross-link structures were investigated for RAAA-tet-K by high-resolution cyclic ion mobility mass spectrometry that identified precursor ion conformers and multiple dissociation products. All sequences were subjected to conformational analysis by Born-Oppenheimer molecular dynamics, and energy analysis by density functional theory calculations with M06-2X/def2qzvpp that provided relative and dissociation energies for several cross-link structural types. The cross-linking reactions were substantially exothermic, driving the efficient conversion of nitrile-imine intermediates to cyclic products. The principal steps in covalent cross-linking involved proton transfer onto the nitrile imine group accompanied by nucleophilic attack by the peptide side-chain and amide groups. Blocking the proton transfer and nucleophile resulted in a loss of cross-linking abilities.

Beneficial Soil Fungus Kills Predatory Nematodes with Dehydropeptides Translocating into the Animal Gut.

Mortierella alpina is a mold fungus that has gained attention for its positive correlation with soil health, plant growth, and applications as a crop biocontrol agent to suppress the threats of nematode pests. To date, the mechanisms underlying the protective traits of M. alpina against these plant parasites have remained elusive. Here we report that abundantly produced peptidic biosurfactants, malpinin A-D, exhibit robust inhibitory activity against nematodes. Nematode assays with malpinin congeners and chemically synthesized analogues revealed that the dehydro amino acid is critical for activity, whereas the N-terminal amino acid residues modulate the lipophilicity. Complementary imaging by fluorescence microscopy and Raman microspectroscopy, using externally fluorescence-labeled, semisynthetic malpinin or a biosynthetically alkyne-tagged probe generated by precursor-directed biosynthesis, visualized the translocation and enrichment of malpinin in the gut of the model nematode Caenorhabditis elegans. Our findings provide valuable insight into the use of M. alpina as a biocontrol agent, emphasizing the ecologically significant role of malpinins as a protective trait. In addition to solving a long-standing riddle, these findings have translational value for applications in agriculture.

Targeting n-myristoyltransferases promotes a pan-Mammarenavirus inhibition through the degradation of the Z matrix protein.

Several Old World and New World Mammarenavirus are responsible for hemorrhagic fever in humans. These enveloped viruses have a bi-segmented ambisense RNA genome that encodes four proteins. All Mammarenavirus identified to date share a common dependency on myristoylation: the addition of the C14 myristic acid on the N-terminal G2 residue on two of their proteins. The myristoylation of the Z matrix protein is required for viral particle budding, while the myristoylation of the signal peptide to the envelope glycoproteins is important for the entry mechanism. Using Mopeia virus as a model, we characterized the interaction of the Z matrix protein with the N-Myristoyltransferases (NMT) 1 and 2, the two enzymes responsible for myristoylation in mammals. While both enzymes were capable to interact with Z, we showed that only NMT1 was important for the production of viral progeny, the endogenous expression of NMT2 being insufficient to make up for NMT1 in its absence. Using the high affinity inhibitors of NMTs, IMP1088 and DDD85646, we demonstrated a strong, dose dependent and specific inhibition at the nanomolar range for all Mammarenavirus tested, including the highly pathogenic Lassa, Machupo, Junin and Lujo viruses. Mechanistically, IMP1088 and DDD85646 blocked the interaction between Z and both NMTs, preventing myristoylation and further viral particle formation, egress and spread. Unexpectedly, we found that the matrix protein devoid of myristate, despite being fully translated, did not accumulate as the other viral proteins in infected cells but was instead degraded in a proteasome- and autophagy-independent manner. These molecules represent a new broad-spectrum class of inhibitors against Mammarenavirus.

Very broad distribution of β sheet registries of the HIV gp41 fusion peptide supports mutational robustness for fusion and infection

HIV, like other membrane-enveloped viruses, has protein spikes that include a fusion peptide (Fp) segment that binds the host cell membrane and plays a critical role in fusion (joining) viral and cell membranes. The HIV Fp is the ~23 N-terminal residues of the gp41 spike protein. Fp adopts intermolecular antiparallel β sheet structure when lipid fraction cholesterol ≈0.3, which is comparable to host cells. Rotational-echo double-resonance NMR was applied to probe the registries (alignments) of adjacent Fp molecules in membrane-bound sheets. The data were fitted to determine quantitative populations, f(t)'s, of individual antiparallel registries indexed by t, the number of residues in the registry. Both wild-type (WT) and fusion-defective V2E Fp sheets have broad but very different registry distributions, each with at least eight populated registries with f(t) > 0.02, and [Formula: see text] and ⟨t⟩V2E = 18.5. The broad WT distribution likely improves mutational robustness for HIV, as Fp is a neutralization epitope of the immune system, and Fp mutations are required for immune evasion during chronic HIV infection. V2E fusion is reduced because longer Fp sheets increase separation between initially apposed membranes. The f(t)WT were well-fitted to free energies that were sums of contributions from sheet length, aligned leucines, and sidechain membrane insertion. The f(t)V2E's were similarly well-fitted except there wasn't the insertion contribution. Relative to V2E, WT fusion is enhanced by deeper membrane insertion of Fp with accompanying greater dislocation of neighboring lipids. This study provides a rare quantitative determination of broad molecular structural distributions by experiment.

Role of chemokines in aging and age-related diseases

Chemokines (chemotactic cytokines) play essential roles in developmental process, immune cell trafficking, inflammation, immunity, angiogenesis, cellular homeostasis, aging, and tumorigenesis. Chemokines also modulate response to immunotherapy, and consequently influence the therapeutic outcome. The mechanisms underlying these processes are accomplished by interaction of chemokines with their cognate cell surface G protein-coupled receptors (GPCRs) and subsequent cellular signaling pathways. Chemokines play crucial role in influencing aging process and age-related diseases across various tissues and organs, primarialy through inflammatory responses (inflammaging), recruitment of macrophages, and orchestrated trafficking of other immune cells. Chemokines are categorized in four distinct groups based on the position and number of the N-terminal cysteine residues; namely, the CC, CXC, CX3C, and (X)C. They mediate inflammatory responses, and thereby considerably impact aging process across multiple organ-systems. Therefore, understanding the underlying mechanisms mediated by chemokines may be of crucial importance in delaying and/or modulating the aging process and preventing age-related diseases. In this review, we highlight recent progress accomplished in understanding the role of chemokines and their cellular signaling pathways involved in aging and age-relaed diseases of various organs. Moreover, we explore potential therapeutic strategies involving chemokines and chemokine receptor antagonists aimed at reducing aging and mitigating age-related diseases. One of the modern methods in this direction involves use of chemokine receptor antagonists and anti-chemokines, which suppress the pro-inflammatory response, thereby helping in resolution of inflammation. Considering the wide-spectrum of functional involvements of chemokines in aging and associated diseases, several clinical trials are being conducted to develop therapeutic approaches using chemokine receptor antagonists to improve life span and promote healthy aging.

Structural Basis for the Pathogenicity of Parkin Catalytic Domain Mutants

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson’s disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several PD mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor-element of parkin (REP) and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin-E2∼Ub complex.

Copper-Mediated Cross-Coupling Selective for Pyroglutamate Post-Translational Modifications.

Pyroglutamate is a cyclic N-terminal post-translational modification that occurs in both proteins and peptide hormones. The prevalence and biological roles of pyroglutamate are little understood, in part due to limited tools to identify, quantify, and manipulate its pyrrolidinone structure. Selective modification of pyroglutamate residues in complex polypeptides may provide unique tools to better understand its biological roles and to allow late-stage diversification of biologically active pyroglutamate-containing sequences. This work describes a copper-catalyzed N-H cross-coupling of unprotected peptides that is selective for N-terminal pyroglutamate residues. The reaction is operationally simple under mild conditions and tolerates all canonical residues. Mechanistic studies point to a key role for a multidentate copper-binding mode of the extended polypeptide structure in delivering the observed reactivity. The reaction allows for direct labeling and identification of a pyroglutamate hormone present in porcine intestinal extracts.

N-terminal cleavage of cyclophilin D boosts its ability to bind F-ATP synthase

Cyclophilin (CyP) D is a regulator of the mitochondrial F-ATP synthase. Here we report the discovery of a form of CyPD lacking the first 10 (mouse) or 13 (human) N-terminal residues (ΔN-CyPD), a protein region with species-specific features. NMR studies on recombinant human full-length CyPD (FL-CyPD) and ΔN-CyPD form revealed that the N-terminus is highly flexible, in contrast with the rigid globular part. We have studied the interactions of FL and ΔN-CyPD with F-ATP synthase at the OSCP subunit, a site where CyPD binding inhibits catalysis and favors the transition of the enzyme complex to the permeability transition pore. At variance from FL-CyPD, ΔN-CyPD binds OSCP in saline media, indicating that the N-terminus substantially decreases the binding affinity for OSCP. We also provide evidence that calpain 1 is responsible for generation of ΔN-CyPD in cells. Altogether, our work suggests the existence of a novel mechanism of modulation of CyPD through cleavage of its N-terminus that may have significant pathophysiological implications.

Aeruginosin 525 (AER525) from Cyanobacterium Aphanizomenon Sp. (KUCC C2): A New Serine Proteases Inhibitor.

Aeruginosins (AERs) are one of the most common classes of cyanobacterial peptides synthesised through a hybrid non-ribosomal peptide synthase/polyketide synthase pathway. They have been found in Microcystis, Nodularia spumigena, Oscillatoria/Plantothrix, and Nostoc. The presence of AER in Aphanizomenon isolated from the Curonian Lagoon was reported for the first time in our previous work. Here, the structure of aeruginosin 525 (AER525), isolated from Aphanizomenon sp. KUCC C2, was characterised based on high-resolution mass spectrometry. This new AER variant shows potent activity against thrombin. It also inhibits trypsin and carboxypeptidase A but has no effect on elastase and chymotrypsin. In terms of the N-terminal residue and biological activity, AER525 displaces some similarity to dysinosins, which belongs to the most potent inhibitors of thrombin among AERs. The findings underline the potential of AER525 as a new anticoagulant agent.

TRPV4-mediated Mechanotransduction Regulates the Differentiation of Valvular Interstitial Cells to Myofibroblasts: Implications for Aortic Stenosis.

As aortic valve stenosis (AVS) progresses, the valve tissue also stiffens. This increase in tissue stiffness causes the valvular interstitial cells (VICs) to transform into myofibroblasts in response. VIC-to-myofibroblast differentiation is critically involved in the development of AVS. Herein, we investigated the role of mechanosensitive Ca 2+ -permeant transient receptor potential vanilloid 4 (Trpv4) channels in matrix stiffness- and transforming growth factor β1 (TGFβ1)-induced VIC-myofibroblast activation. We confirmed Trpv4 functionality in primary mouse wild-type VICs compared to Trpv4 null VICs using live Ca 2+ influx detection during application of its selective agonist and antagonist. Using physiologically relevant hydrogels of varying stiffness that respectively mimic healthy or diseased aortic valve tissue stiffness, we found that genetic ablation of Trpv4 blocked matrix stiffness- and TGFβ1-induced VIC-myofibroblast activation as determined by changes in morphology, alterations of expression of α-smooth muscle actin, and modulations of F-actin generation. Our results showed that N-terminal residues 30-130 in Trpv4 were crucial for cellular force generation and VIC-myofibroblast activation, while deletion of residues 1-30 had no noticeable negative effect on these processes. Collectively, these data suggest a differential regulatory role for Trpv4 in stiffness/TGFβ1-induced VIC-myofibroblast activation. Our data further showed that Trpv4 regulates stiffness/TGFβ1-induced PI3K-AKT activity that is required for VIC-myofibroblast differentiation and cellular force generation, suggesting a mechanism by which Trpv4 activity regulates VIC-myofibroblast activation. Altogether, these data identify a novel role for Trpv4 mechanotransduction in regulating VIC-myofibroblast activation, implicating Trpv4 as a potential therapeutic target to slow and/or reverse AVS development.

Formylation facilitates the reduction of oxidized initiator methionines

Within a cell, protein-bound methionines can be chemically or enzymatically oxidized, and subsequently reduced by methionine sulfoxide reductases (Msrs). Methionine oxidation can result in structural damage or be the basis of functional regulation of enzymes. In addition to participating in redox reactions, methionines play an important role as the initiator residue of translated proteins where they are commonly modified at their α-amine group by formylation or acetylation. Here, we investigated how formylation and acetylation of initiator methionines impact their propensity for oxidation and reduction. We show that in vitro, N-terminal methionine residues are particularly prone to chemical oxidation and that their modification by formylation or acetylation greatly enhances their subsequent enzymatic reduction by MsrA and MsrB. Concordantly, in vivo ablation of methionyl-tRNA formyltransferase (MTF) in Escherichia coli increases the prevalence of oxidized methionines within synthesized proteins. We show that oxidation of formylated initiator methionines is detrimental in part because it obstructs their ensuing deformylation by peptide deformylase (PDF) and hydrolysis by methionyl aminopeptidase (MAP). Thus, by facilitating their reduction, formylation mitigates the misprocessing of oxidized initiator methionines.

Effect of Surface-Immobilized States of Antimicrobial Peptides on Their Ability to Disrupt Bacterial Cell Membrane Structure.

Antimicrobial peptide (AMP) surfaces are widely used to inhibit biofilm formation and bacterial infection. However, endpoint-immobilized AMPs on surfaces are totally different from free-state AMPs due to the constraints of the surface. In this work, the interactions between AMPs and bacterial cell membranes were analyzed through coarse-grained molecular dynamics and all-atom molecular dynamics simulations. This AMP disrupted membrane structure by altering the thickness and curvature of the membrane. Furthermore, the effect of surface-immobilized states of AMPs on their ability to disrupt membrane structure was revealed. The immobilized AMPs in the freeze-N system could bind to the membrane and disrupt the membrane structure through electrostatic forces between positively charged N-terminal amino acid residues and the negatively charged membrane, while the immobilized AMPs in the freeze-C system were repelled. The results will aid in the rational design of new AMP surfaces with enhanced efficacy and stability.

Identification of the N-terminal residues responsible for the differential microdomain localization of CYP1A1 and CYP1A2

The endoplasmic reticulum (ER) is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains characterized by more fluidity. Rabbit CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera containing the first 109 amino acids of CYP1A1 showed altered microdomain localization. The goal of this study was to identify specific residues responsible for CYP1A microdomain localization. Thus, CYP1A2 chimeras containing substitutions from homologous regions of CYP1A1 were expressed in HEK 293T/17 cells, and the localization was examined after solubilization with Brij 98. A CYP1A2 mutant with the three amino acids from CYP1A1 (VAG) at positions 27-29 of CYP1A2 was generated that showed a distribution pattern similar to those of CYP1A1/1A2 chimeras containing both the first 109 amino acids and the first 31 amino acids of CYP1A1 followed by remaining amino acids of CYP1A2. Similarly, the reciprocal substitution of three amino acids from CYP1A2 (AVR) into CYP1A1 resulted in a partial redistribution of the chimera into ordered microdomains. Molecular dynamic simulations indicate that the positive charges of the CYP1A1 and CYP1A2 linker regions between the N-termini and catalytic domains resulted in different depths of immersion of the N-termini in the membrane. The overlap of the distribution of positively charged residues in CYP1A2 (AVR) and negatively charged phospholipids was higher in the ordered than disordered microdomain. These findings identify three residues in the CYP1A N-terminus as a novel microdomain-targeting motif of the P450s and provide a mechanistic explanation for the differential microdomain localization of CYP1A.

Split MutT prevents the mutator phenotype of mutT-deficient Escherichia coli

BackgroundThe Escherichia coli MutT (NudA) protein catalyzes the hydrolysis of an oxidized form of dGTP, 8-oxo-7,8-dihydro-dGTP (8-hydroxy-dGTP), and the spontaneous mutation frequency is elevated in E. coli cells deficient in the mutT gene.ResultsA split MutT, comprising the N-terminal (residues 1–95) and C-terminal (residues 96–129) peptides, was designed based on the known tertiary structure and linker insertion mutagenesis experiments. The mutator phenotype was complemented when the two peptides were separately expressed in mutT E. coli cells.ConclusionsThese results indicated that this split MutT functions as a nucleotide pool sanitization enzyme in vivo.

Mechanisms underlying TRPV4-mediated regulation of miR-146a expression.

Persistent inflammation is a major contributor in the development of various inflammatory diseases like atherosclerosis. Our study investigates how transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, interacts with microRNA-146a (miR-146a), within the context of inflammation and atherosclerosis. Micro-RNAs play a critical role in controlling gene expression, and miR-146a is notable for its anti-inflammatory actions. TRPV4 is activated by diverse soluble and mechanical stimuli, and often associated with inflammatory responses in various diseases. Here, we find that TRPV4 negatively regulates miR-146a expression in macrophages, especially following stimulation by lipopolysaccharides or alterations in matrix stiffness. We show that in atherosclerosis, a condition characterized by matrix stiffening, TRPV4 decreases miR-146a expression in aortic tissue macrophages. We find that TRPV4's impact on miR-146a is independent of activation of NFκB, Stat1, P38, and AKT, but is rather mediated through a mechanism involving histone deacetylation instead of DNA methylation at the miR-146a promoter site. Furthermore, we show that N-terminal residues 1 to 130 in TRPV4 is essential in suppression of miR-146a expression in LPS-stimulated macrophages. Altogether, this study identifies a regulatory mechanism of miR-146a expression by TRPV4 which may open new potential therapeutic strategies for managing inflammatory diseases.

New Supramolecular Hydrogels Based on Diastereomeric Dehydrotripeptide Mixtures for Potential Drug Delivery Applications.

Self-assembly of peptide building blocks offers unique opportunities for bottom-up preparation of exquisite nanostructures, nanoarchitectures, and nanostructured bulk materials, namely hydrogels. In this work we describe the synthesis, characterization, gelation, and rheological properties of new dehydrotripeptides, Cbz-L-Lys(Cbz)-L,D-Asp-∆Phe-OH and (2-Naph)-L-Lys(2-Naph)-L,D-Asp-∆Phe-OH, containing a N-terminal lysine residue Nα,ε-bis-capped with carboxybenzyl (Cbz) and 2-Naphthylacetyl (2-Naph) aromatic moieties, an aspartic acid residue (Asp), and a C-terminal dehydrophenylalanine (∆Phe) residue. The dehydrotripeptides were obtained as diastereomeric mixtures (L,L,Z and L,D,Z), presumably via aspartimide chemistry. The dehydrotripeptides afforded hydrogels at exceedingly low concentrations (0.1 and 0.04 wt%). The hydrogels revealed exceptional elasticity (G' = 5.44 × 104 and 3.43 × 106 Pa) and self-healing properties. STEM studies showed that the diastereomers of the Cbz-capped peptide undergo co-assembly, generating a fibrillar 3D network, while the diastereomers of the 2-Naph-capped dehydropeptide seem to undergo self-sorting, originating a fibril network with embedded spheroidal nanostructures. The 2-Naph-capped hydrogel displayed full fast recovery following breakup by a mechanical stimulus. Spheroidal nanostructures are absent in the recovered hydrogel, as seen by STEM, suggesting that the mechanical stimulus triggers rearrangement of the spheroidal nanostructures into fibers. Overall, this study demonstrates that diastereomeric mixtures of peptides can be efficacious gelators. Importantly, these results suggest that the structure (size, aromaticity) of the capping group can have a directing effect on the self-assembly (co-assembly vs. self-sorting) of diastereomers. The cytotoxicity of the newly synthesized gelators was evaluated using human keratinocytes (HaCaT cell line). The results indicated that the two gelators exhibited some cytotoxicity, having a small impact on cell viability. In sustained release experiments, the influence of the charge on model drug compounds was assessed in relation to their release rate from the hydrogel matrix. The hydrogels demonstrated sustained release for methyl orange (anionic), while methylene blue (cationic) was retained within the network.

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity

Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.

Folding of N-terminally acetylated α-synuclein upon interaction with lipid membranes

α-Synuclein (α-syn) is an abundant presynaptic neuronal protein whose aggregation is strongly associated with Parkinson’s disease. It has been proposed that lipid membranes significantly affect α-syn’s aggregation process. Extensive studies have been conducted to understand the interactions between α-syn and lipid membranes and have demonstrated that the N-terminus plays a critical role. However, the dynamics of the interactions and the conformational transitions of the N-terminus of α-syn at the atomistic scale details are still highly desired. In this study, we performed extensive enhanced sampling molecular dynamics simulations to quantify the folding and interactions of wild-type and N-terminally acetylated α-syn when interacting with lipid structures. We found that N-terminal acetylation significantly increases the helicity of the first few residues in solution or when interacting with lipid membranes. The observations in simulations showed that the binding of α-syn with lipid membranes mainly follows the induced-fit model, where the disordered α-syn binds with the lipid membrane through the electrostatic interactions and hydrophobic contacts with the packing defects; after stable insertion, N-terminal acetylation promotes the helical folding of the N-terminus to enhance the anchoring, thus increasing the binding affinity. We have shown the critical role of the first N-terminal residue methionine for recognition and anchoring to the negatively charged membrane. Although N-terminal acetylation neutralizes the positive charge of Met1 that may affect the electrostatic interactions of α-syn with membranes, the increase in helicity of the N-terminus should compensate for the binding affinity. This study provides detailed insight into the folding dynamics of α-syn’s N-terminus with or without acetylation in solution and upon interaction with lipids, which clarifies how the N-terminal acetylation regulates the affinity of α-syn binding to lipid membranes. It also shows how packing defects and electrostatic effects coregulate the N-terminus of α-syn folding and its interaction with membranes.

A novel cystatin in Psoroptes ovis var. cuniculi: molecular characterization, serodiagnostic potential, and its anti-inflammatory property on rabbit peripheral blood mononuclear cells

BackgroundThe ectoparasite Psoroptes ovis var. cuniculi causes substantial economic losses to the global rabbit industry. Currently, microscopy for identifying Psoroptes mite in skin scrapings, as the “diagnosis gold standard,” remains a challenge owing to its poor sensitivity in detecting low-level and/or early stage mite infestations. Additionally, Psoroptes infestations rapidly trigger cutaneous inflammation, thus the mites might produce some molecules to deal with the harmful effects of inflammation for their long-time survival on the host skin, but these molecules are still mostly unknown.MethodsTo seek a sensitive diagnostic method and illuminate the new antiinflammatory molecules, we characterized a novel cystatin of P. ovis var. cuniculi (PsoCys) using bioinformatics and molecular biology methods.ResultsThe results showed that PsoCys comprised the classical features of the type II cystatin superfamily including an N-terminal glycine residue, a central QXVXG motif, and a C-terminal LW motif. In mixed stages of mites, the transcription level of PsoCys was significantly higher in “fed” mites than in “starved” mites (P < 0.001), and among the different life-cycle stages of “fed” mites, the expression of PsoCys was higher in adult males than in larva, nymph, and adult females (P < 0.001). The established indirect ELISA based on recombinant PsoCys (rPsoCys-iELISA) presented 95.4% sensitivity and 95.7% specificity. The area under the receiver operating characteristic curve (AUC) for this method was 0.991, indicating its excellent diagnostic performance. Moreover, rPsoCys-iELISA had advantages over microscopy for detecting low-level and/or early stage mite infestations (90% versus 40% in artificial infestation cases at 3 weeks post-infestation; 61.9% versus 22.6% in clinical cases). In addition, rPsoCys could inhibit the activity of papain and cathepsin B in vitro, and significantly suppressed mRNA levels of toll-like receptors (TLR 1, 2, 4, and 6) and downstream molecules (NF-κB, p38, MyD88, IL-10, and IFN-γ) in LPS-stimulated rabbit PBMCs, indicating its anti-inflammatory property.ConclusionsOur findings indicated that PsoCys was a novel type II cystatin of Psoroptes mites, and it served as a potential serological diagnostic antigen for detecting low-level and/or early stage mite infestations, as well as a novel anti-inflammatory molecule of Psoroptes mites.Graphical abstract

N-degron pathways

An N-degron is a degradation signal whose main determinant is a “destabilizing” N-terminal residue of a protein. Specific N-degrons, discovered in 1986, were the first identified degradation signals in short-lived intracellular proteins. These N-degrons are recognized by a ubiquitin-dependent proteolytic system called the Arg/N-degron pathway. Although bacteria lack the ubiquitin system, they also have N-degron pathways. Studies after 1986 have shown that all 20 amino acids of the genetic code can act, in specific sequence contexts, as destabilizing N-terminal residues. Eukaryotic proteins are targeted for the conditional or constitutive degradation by at least five N-degron systems that differ both functionally and mechanistically: the Arg/N-degron pathway, the Ac/N-degron pathway, the Pro/N-degron pathway, the fMet/N-degron pathway, and the newly named, in this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these systems and the expanded terminology that now encompasses the entire gamut of known N-degron pathways.