Disulfide Bridges Research Articles

Study on the Analgesic Activity of Peptide from Conus achates.

As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 μg/kg) and Trx-Ac6.4 (20, 40, 80 μg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.

Development of a System for Biosynthesis, Isolation and Purification of Holoform of Recombinant Human Neuroglobin and Its Characteristics

An efficient system for the biosynthesis, isolation and purification of recombinant human neuroglobin has been developed and optimized, which makes it possible to produce protein in quantities sufficient to study its properties. According to UV-visible, IR-, CD-, and NMR spectroscopy data, recombinant neuroglobin is a structured protein in the holoform state. The data of chromato-mass-spectrometric analysis made it possible to conclude that there is a correctly formed disulfide bond in the structure of the oxidized form of the protein. Using Raman and surface-enhanced Raman spectroscopy with laser excitation at 532 nm, it was shown that heme in the reduced and oxidized forms of neuroglobin has vibrational degrees of freedom typical of b-type hemes, and the iron atom is six-coordinated. Using Raman spectroscopy with laser excitation at 633 nm, it was found that reduced –SH-groups were present in reduced neuroglobin, while in oxidized neuroglobin disulfide bridge was formed. The results obtained serve as the basis for detailed studies of the mechanism of the functioning of neuroglobin as a neuroprotector, in particular, during its interaction with oxidized cytochrome c, which is released from mitochondria in violation of their functioning and/or morphology.

Metabotropic glutamate receptors in GtoPdb v.2023.1

Metabotropic glutamate (mGlu) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Metabotropic Glutamate Receptors [351]) are a family of G protein-coupled receptors activated by the neurotransmitter glutamate [140]. The mGlu family is composed of eight members (named mGlu1 to mGlu8) which are divided in three groups based on similarities of agonist pharmacology, primary sequence and G protein coupling to effector: Group-I (mGlu1 and mGlu5), Group-II (mGlu2 and mGlu3) and Group-III (mGlu4, mGlu6, mGlu7 and mGlu8) (see Further reading).Structurally, mGlu are composed of three juxtaposed domains: a core G protein-activating seven-transmembrane domain (TM), common to all GPCRs, is linked via a rigid cysteine-rich domain (CRD) to the Venus Flytrap domain (VFTD), a large bi-lobed extracellular domain where glutamate binds. mGlu form constitutive dimers, cross-linked by a disulfide bridge. The structures of the VFTD of mGlu1, mGlu2, mGlu3, mGlu5 and mGlu7 have been solved [200, 275, 268, 403]. The structure of the 7 transmembrane (TM) domains of both mGlu1 and mGlu5 have been solved, and confirm a general helical organisation similar to that of other GPCRs, although the helices appear more compacted [88, 433, 62]. Recent advances in cryo-electron microscopy have provided structures of full-length mGlu receptor homodimers [217, 191] and heterodimers [91]. Studies have revealed the possible formation of heterodimers between either group-I receptors, or within and between group-II and -III receptors [89]. First characterised in transfected cells, co-localisation and specific pharmacological properties suggest the existence of such heterodimers in the brain [270, 440, 145, 283, 259, 218]. Beyond heteromerisation with other mGlu receptor subtypes, increasing evidence suggests mGlu receptors form heteromers and larger order complexes with class A GPCRs (reviewed in [140]). The endogenous ligands of mGlu are L-glutamic acid, L-serine-O-phosphate, N-acetylaspartylglutamate (NAAG) and L-cysteine sulphinic acid. Group-I mGlu receptors may be activated by 3,5-DHPG and (S)-3HPG [30] and antagonised by (S)-hexylhomoibotenic acid [235]. Group-II mGlu receptors may be activated by LY389795 [269], LY379268 [269], eglumegad [354, 434], DCG-IV and (2R,3R)-APDC [355], and antagonised by eGlu [170] and LY307452 [425, 105]. Group-III mGlu receptors may be activated by L-AP4 and (R,S)-4-PPG [130]. An example of an antagonist selective for mGlu receptors is LY341495, which blocks mGlu2 and mGlu3 at low nanomolar concentrations, mGlu8 at high nanomolar concentrations, and mGlu4, mGlu5, and mGlu7 in the micromolar range [185]. In addition to orthosteric ligands that directly interact with the glutamate recognition site, allosteric modulators that bind within the TM domain have been described. Negative allosteric modulators are listed separately. The positive allosteric modulators most often act as ‘potentiators’ of an orthosteric agonist response, without significantly activating the receptor in the absence of agonist.

CogiTx1: A novel subtilisin A inhibitor isolated from the sea anemone Condylactis gigantea belonging to the defensin 4 protein family

Subtilisin-like enzymes are recognized as key players in many infectious agents. In this context, its inhibitors are very valuable molecular lead compounds for structure based drug discovery and design. Marine invertebrates offer a great source of bioactive molecules, including protease inhibitors. In this work, we describe a new subtilisin inhibitor, from the sea anemone Condylactis gigantea (CogiTx1). CogiTx1 was purified using a combination of cation exchange chromatography, size exclusion chromatography and RP-HPLC chromatography. CogiTx1 it is a protein with 46 amino acid residues, with 4970.44 Da and three disulfide bridges. Is also able to inhibit subtilisin-like enzymes and pancreatic elastase. According to the amino acid sequence, it belongs to the defensin 4 family of proteins. The sequencing showed that CogiTx1 has an amidated C-terminal end, which was confirmed by the presence of the typical –XGR signal for amidation in the protein sequence deduced from the cDNA. This modification was described at protein level for the first time in this family of proteins. CogiTx1 is the first subtilisin inhibitor from the defensin 4 family and accordingly it has a folding consisting primarily in beta-strands in agreement with the analysis by CD and 3D modelling. Therefore, future in-depth functional studies may allow a more detailed characterization and will shed light on structure-function properties.

Structural dynamics and mechanistic action guided engineering of lipolytic enzymes.

Lipases have been established as important biocatalysts in several industrial applications, owing to their diverse substrate specificity. The availability of data on three-dimensional crystal structures for various lipases offers an opportunity for modulating their structural and functional aspects to design and engineer better versions of lipases. With the aim of investigating the structural components governing the extremophilic behavior of lipases, structural analysis of microbial lipases was performed using advanced bioinformatics and molecular dynamics simulation approaches. In sequences and functionally distinct alkaliphilic and thermophilic lipases were investigated for their functional properties to understand the distinguishing features of their structures. The alkaliphilic lipase from Bacillus subtilis (LipA) showed conformational changes in the loop region Ala132-Met137, subsequently, the active site residue His156 shows two conformations, toward the active site nucleophilic residues Ser77 and away from the Ser77. Interestingly, the active site of LipA is more solvent-exposed and can be correlated with the adoption of an open conformation which might extend and expose the active site region to solvents during the catalysis process. Furthermore, the MD simulation of thermophilic lipase from marine Streptomyces (MAS1) revealed the role of N- and C-terminal regions with disulfide bridges and identified a metal ion binding site that facilitates the enzyme stability. The novel thermo-alkaliphilic lipase can be designed to integrate the stability features of MAS1 into the alkaliphilic LipA. These structural-level intrinsic characteristics can be used for lipase engineering to amend the lipase activity and stability as per the requirements of the industrial processes.

TRAP1 S-nitrosylation as a model of population-shift mechanism to study the effects of nitric oxide on redox-sensitive oncoproteins

S-nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S-nitrosothiol (SNO) adduct. S-nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S-nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S-nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S-nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S-nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows (https://github.com/ELELAB/SNO_investigation_pipelines) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S-nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S-nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field.

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation.

Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETaseCD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETaseCD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETaseCD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (Tm) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

SynDLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial dynamin-like proteins are still poorly investigated. SynDLP, the dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. In addition to typical GD-GD contacts, such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids independent of nucleotides. The structural characteristics of SynDLP oligomers suggest it to be the closest known bacterial ancestor of eukaryotic dynamin.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

BackgroundThe von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. ObjectiveTo determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. MethodsWe combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. ResultsWe show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. ConclusionsOur data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

Mucoadhesive thermogel platform for treating anterior ocular conditions.

A platform mucoadhesive and thermogelling eyedrop was developed for application to the inferior fornix for the treatment of various anterior segment ocular conditions. The poly(n-isopropylacrylamide) polymers (pNIPAAm), containing a disulfide bridging monomer, were crosslinked with chitosan to yield a modifiable, mucoadhesive, and natively degradable thermogelling system. Three different conjugates were studied including a small molecule for treating dry eye, an adhesion peptide for modeling delivery of peptides/proteins to the anterior eye, and a material property modifier to create gels with different rheologic characteristics. Based on the conjugate used, different material properties such as solution viscosity and lower critical solution temperature (LCST) were produced. In addition to releasing the conjugates through disulfide bridging with ocular mucin, the thermogels were shown to deliver atropine, with 70%-90% being released over 24-h, depending on the formulation studied. The results illustrate that these materials can deliver multiple therapeutic payloads at one time and release them through various mechanisms. Finally, the safety and tolerability of the thermogels was demonstrated both in vitro and in vivo. The gels were instilled into the inferior fornix of rabbits and were shown to not produce any adverse effects over 4 days. These materials were demonstrated to be highly tunable, creating a platform that could be easily modified to deliver various therapeutic agents to treat a multitude of ocular diseases and have the potential to be an alternative to conventional eyedrops.

Abstract 3877: Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer

Abstract The estrogen receptor-positive (ER+) subtype accounts for 70% of breast cancer, and endocrine therapies that target ER are effective in the early-stage clinical setting. However, endocrine resistance is ubiquitous in late-stage metastatic breast cancer (MBC). The chief mechanism is the acquisition of ER mutations (ERmuts) in 25% of endocrine-resistant tumors; ERmuts are strongly resistant to aromatase inhibitor (AI) endocrine therapy. We previously identified four novel acquired activating mutations in human epidermal growth factor receptor 2 (HER2) by whole exome sequencing (WES) of paired ER+ MBC in a clinical cohort. These HER2 mutations (HER2muts) were enriched in multiple resistant tumors, and like ERmuts, were resistant to AIs. In contrast to ERmuts, these HER2muts also conferred complete resistance to all other classes of endocrine therapy, as well as cross-resistance to combination therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors; the latter is standard-of-care in the refractory setting in ER+ MBC. At the same time, these HER2muts had variable responses to HER2-targeting therapies used in HER2+ breast cancer. Thus, HER2muts represent only the second validated mechanism of clinical endocrine resistance in ER+ MBC, accounting for ~8% of cases, and that is therapeutically intractable. Systematic phenotypic characterization of HER2muts in ER+ MBC is a prerequisite for effective targeting of this class of resistance. We used cBioPortal to identify >20 novel HER2 variants of unknown significance (VUS) that were frequent pan-cancer, in ER+ breast tumors, or annotated in a domain-restricted HER2 saturation mutagenesis screen, and that had not been previously characterized in ER+ MBC. We also identified a novel HER2 VUS by WES in an ER+/HER2+ tumor with resistance to HER2 therapy, from a clinical cohort of breast cancer patients. We phenotypically characterized all VUS through clinically comprehensive targeted therapy viability assays, and downstream activation assays. In sum, we identified seven novel activating HER2muts, spanning domains, with clinically important phenotypes in ER+ MBC. We found these HER2muts also conferred complete resistance to all classes of endocrine therapies, and cross-resistance to CDK4/6 inhibition. While novel HER2muts had variable responses to HER2-targeting therapies, they could be endocrine re-sensitized by combination with an irreversible tyrosine kinase inhibitor. Lastly, we report that constitutive HER2 homodimerization via reduction-sensitive disulfide bridges that occur as a consequence of mutations in the extracellular and juxtamembrane domains is a more common mechanism of HER2 hyperactivation than previously appreciated. This finding has unique functional consequences, and provides important structure-function insight into HER2 biology, beyond its immediate clinical ramifications in ER+ MBC. Citation Format: Nelson Vicente Guevara, Zirui Zhu, Esha Jain, Jennifer Kavran, Selime Arslan, Nikhil Wagle, Utthara Nayar. Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3877.

Vanillin-Based Epoxy Vitrimers: Looking at the Cystamine Hardener from a Different Perspective.

Epoxy vitrimers encompass many advantages compared to traditional epoxy materials such as recyclability, repairability, and reprocessability. These properties are induced by the incorporation of dynamic reversible covalent bonds. Recently, the incorporation of aromatic disulfide bridges that are dynamic has expanded the development of new eco-friendly epoxy materials. Herein, we studied a bio-based aliphatic disulfide based on cystamine as a hardener with a vanillin-derived bio-sourced epoxy to prepare fully bio-based epoxy vitrimers. This article provides a comparative study between cystamine and an aromatic disulfide benchmark hardener issued from petrol resources. This work demonstrated that the presence of this aliphatic hardener has a significant influence not only on the reactivity, but most importantly on the resulting dynamic properties. An interesting yet counterintuitive accelerating effect of the dynamic exchanges was clearly demonstrated with only 2 to 20% of molar fraction of cystamine added to the aromatic disulfide formulation. A similar glass transition was obtained compared to the purely aromatic analogue, but relaxation times were decreased by an order of magnitude.

Disulfide-Bridged Dendritic Organosilicas-Based Biodegradable Molecularly Imprinted Polymers for Multiple Targeting and pH/Redox-Responsive Drug Release toward Chemical/Photodynamic Synergistic Tumor Therapy.

In this study, a sialic acid (SA) and transferrin (TF) imprinted biodegradable disulfide bridging organosilicas-based drug delivery system (SS-DMONS/DOX-Ce6@MIPs) for targeted cancer therapy is constructed, for the first time. Disulfide bridged dendritic mesoporous organosilicas nanoparticles (SS-DMONs) not only enhance drug loading as the drug repository, but also provide enough specific surface area for the molecular imprinting shell to expose more degradation and imprinted sites on the surface. In addition, SS can be disturbed in a highly reducing tumor microenvironment to achieve degradation. The biodegradable imprinting film, prepared with customized 2-amino-N-(3,4-dihydroxyphenethyl)-3-mercaptopropanamide and 4-mercaptophenylboronic acid as functional monomers, endows SS-DMONs with active targeting capacity, and responsive drug release through degradation under acidic and highly reductive tumor microenvironment. SS-DMONS/DOX-Ce6@MIPs after binding of TF can target tumor cells actively through multiple interactions, including the affinity between antigen and antibody, and the specific recognition between molecularly imprinted polymers and template molecules. Under laser irradiation the loaded chlorin e6 (Ce6) that can produce toxic reactive oxygen, combined with the doxorubicin (DOX), achieves chemical/photodynamic synergistic anticancer effects. SS-DMONS/DOX-Ce6@MIPs present excellent tumor targeting and dual-responsive drug release, which provides an effective strategy for chemical/photodynamic antitumor therapy.

A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity.

Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin's receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.

Identification of biomarkers in diabetic nails by Raman spectroscopy

BackgroundThe growth of Diabetes Mellitus (DM) is a serious public health issue which is more prevalent in developing countries. The main problems related to DM are the gradual changes in the structural and functional integrity of tissues caused by hyperglycemia, which calls for early diagnosis and periodic monitoring exams. Recent studies suggest that the quality of the nail plate has great potential to assess the secondary complications of DM. Hence, this study aimed to determine the biochemical characteristics of the nails of individuals with DM2 by Raman confocal spectroscopy (CRS). MethodsWe collected fragments from the distal region of the fingernails of 30 healthy volunteers and 30 volunteers with DM2. The samples were analyzed by CRS (Xplora - Horiba) coupled to a 785 nm laser. ResultsAlterations in different biochemical components, such as proteins, lipids, amino acids, and final agents of advanced glycation, and alterations in the disulfide bridges, which are important in stabilizing keratin in nails were identified. ConclusionThe spectral signatures and new DM2 markers in nails were identified. Therefore, the possibility of acquiring biochemical information by evaluating the nails of diabetics, a simple and easily acquired material associated with the CRS technique, may allow health complications to be detected quickly.

LAMP GENE- Structure, Function and Involvement in Cancer

Abstract: The LAMP1 gene in humans produces the lysosomal-associated membrane protein 1 (LAMP-1), also known as lysosome-associated membrane glycoprotein 1 and CD107a (Cluster of Differentiation 107a). The human LAMP1 gene is found in region 3, band 4 of chromosome 13's long arm (q). These glycoproteins, which are typically found across lysosomal membranes, have a long, heavily glycosylated end on the luminal side of the membrane with N-linked carbon chains and a short, cytoplasm-expose C-terminal tail. A structure resembling a hinge exists in the extracytoplasmic region, and it is capable of forming disulphide bridges that are similar to those found in human immunoglobulin A Cancer cells frequently express LAMP1 and LAMP2 on their cell surfaces, especially tumours with a high propensity to metastasize, such melanoma and colon cancer, which has been found to be correlated with the propensity to metastasize. Danon disease, which is brought on by a change (mutation) in the LAMP2 gene. Currently, case reports and databases have identified approximately 160 distinct LAMP2 gene variants that have the potential to cause Danon disease. The prognosis has been shown to be most adversely affected by mutations that result in the total lack of the LAMP2 protein.

Surfactant enhances anaerobic fermentative hydrogen sulfide production: Changes in sulfur-containing organics structure and microbial community

The presence of surfactants in waste activated sludge (WAS) system is generally regarded as beneficial to sludge treatment such as enhancing sludge dewatering and improving value-added fermentation products generation. However, in this study, it was firstly found that sodium dodecylbenzene sulfonate (SDBS, a typical surfactant) obviously increased toxic hydrogen sulfide (H2S) gas production from WAS anaerobic fermentation at environmentally relevant concentrations. Experimental results showed that H2S production from WAS significantly increased from 53.24 × 10−3 to 111.25 × 10−3 mg/g volatile suspended solids (VSS) when SDBS level increased from 0 to 30 mg/g total suspended solid (TSS). It was found that SDBS presence destroyed WAS structure and enhanced sulfur containing organics release. SDBS reduced the proportion of α-helix structure, damaged disulfide bridges and protein conformation, and effectively destroyed protein structure. SDBS promoted sulfur containing organics degradation and provided more readily hydrolyzed micro-molecule organics for sulfide production. Microbial analysis showed that SDBS addition enhanced the abundance of functional genes encoding protease, ATP-binding cassette transporters, and amino acids lyase, enhanced the activities and abundance of hydrolytic microbes, thus increased sulfide production from the hydrolysis of sulfur containing organics. Compared with the control, 30 mg/g TSS SDBS increased organic sulfurs hydrolysis and amino acids degradation by 47.1 % and 63.5 %, respectively. Key genes analysis further showed that SDBS addition promoted sulfate transport system and dissimilatory sulfate reduction. SDBS presence also lowered fermentation pH, promoted the chemical equilibrium transformation of sulfide, thus increased H2S gas release.

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

Antibody–drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Pmu1a, a novel spider toxin with dual inhibitory activity at pain targets hNaV 1.7 and hCaV 3 voltage-gated channels.

Venom-derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage-gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV 1.7 and hCaV 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay-guided HPLC fractionation revealed a 36-amino acid peptide with three disulfide bridges named μ/ω-theraphotoxin-Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized, and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV 1.7 and hCaV 3. NMR structure determination of Pmu1a shows an inhibitor cystine knot (ICK) fold that is characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV 3.2 and hNaV 1.7 voltage-gated channels.