Disulphide Bond Formation Research Articles

Insulin gene mutations linked to permanent neonatal diabetes mellitus in Indian population.

Neonatal diabetes mellitus (NDM) is a rare monogenic disorder of pancreatic beta cell mass and/or function. In the present study we aimed to evaluate the INS gene mutations in a cohort of children with Permanent Neonatal Diabetes Mellitus (PNDM) and to explore the clinical and genetic characteristics of PNDM caused by INS mutations. Direct sequencing of all exons of INS genes was carried out in 189 children with PNDM. Clinical and biochemical data were collected and correlated. The pathogenicity of mutations was determined based on the American College of Medical Genetics and Genomics and Association of Medical Pathology guidelines. Two novel mutations (His34Pro, Leu35Met) in a compound heterozygous state and seven known mutations (Gly32Ser, Phe48Cys, Arg89Cys, Cys96Tyr, Ser98Ile, Try108Asp and Cys109Phe) in the INS gene were identified in 8 patients out of the total of 189 PNDM children studied. Four mutations were involved in defects with disulphide bond formation and hence were in crucial regions of the gene. All the mutations were de novo in origin. This is the first comprehensive study from India to investigate the insulin gene mutations in PNDM and to show that INS gene mutations also contribute to the causation of PNDM.

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration.

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

Post-translational modifications in tumor-associated carbonic anhydrases.

Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.

DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E.coli T7 system.

SummarySecreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post‐translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide‐rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

Cellular Response against Oxidative Stress, a Novel Insight into Lupus Nephritis Pathogenesis.

The interaction of reactive oxygen species (ROS) with lipids, proteins, nucleic acids and hydrocarbonates promotes acute and chronic tissue damage, mediates immunomodulation and triggers autoimmunity in systemic lupus erythematous (SLE) patients. The aim of the study was to determine the pathophysiological mechanisms of the oxidative stress-related damage and molecular mechanisms to counteract oxidative stimuli in lupus nephritis. Our study included 38 SLE patients with lupus nephritis (LN group), 44 SLE patients without renal impairment (non-LN group) and 40 healthy volunteers as control group. In the present paper, we evaluated serum lipid peroxidation, DNA oxidation, oxidized proteins, carbohydrate oxidation, and endogenous protective systems. We detected defective DNA repair mechanisms via 8-oxoguanine-DNA-glycosylase (OGG1), the reduced regulatory effect of soluble receptor for advanced glycation end products (sRAGE) in the activation of AGE-RAGE axis, low levels of thiols, disulphide bonds formation and high nitrotyrosination in lupus nephritis. All these data help us to identify more molecular mechanisms to counteract oxidative stress in LN that could permit a more precise assessment of disease prognosis, as well as developing new therapeutic targets.

Chemokines and Pain in the Trigeminal System.

Chemotactic cytokines or chemokines are a large family of secreted proteins able to induce chemotaxis. Chemokines are categorized according to their primary amino acid sequence, and in particular their cysteine residues that form disulphide bonds to maintain the structure: CC, CXC, CX3C, and XC, in which X represents variable amino acids. Among their many roles, chemokines are known to be key players in pain modulation in the peripheral and central nervous systems. Thus, they are promising candidates for novel therapeutics that could replace current, often ineffective treatments. The spinal and trigeminal systems are intrinsically different beyond their anatomical location, and it has been suggested that there are also differences in their sensory mechanisms. Hence, understanding the different mechanisms involved in pain modulation for each system could aid in developing appropriate pharmacological alternatives. Here, we aim to describe the current landscape of chemokines that have been studied specifically with regard to trigeminal pain. Searching PubMed and Google Scholar, we identified 30 reports describing chemokines in animal models of trigeminal pain, and 15 reports describing chemokines involved in human pain associated with the trigeminal system. This review highlights the chemokines studied to date at different levels of the trigeminal system, their cellular localization and, where available, their role in a variety of animal pain models.

MO076REGULATION OF DIFFERENTIAL CELLULAR RESPONSE TO OXIDATIVE STIMULI IN SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract Background and Aims Interaction of reactive oxygen species (ROS) with lipids, proteins, nucleic acids and hydro carbonates promotes acute and chronic tissue damage, mediates immunomodulation and triggers autoimmunity in systemic lupus erythematous (SLE) patients. The aim of the study was to determine the pathophysiological mechanisms of the oxidative stress-related damage and molecular mechanisms to counteract oxidative stimuli in lupus nephritis. Method Our study included 82 volunteers with SLE: 38 SLE volunteers with lupus nephritis (LN group) and 44 SLE volunteers without renal impairment (non-LN group) and a control group of 40 healthy volunteers. LN was diagnosed by histological exam (optic microscopy, electronic microscopy and immunofluorescence). Disease activity was measured by systemic SLE disease activity index (SLEDAI), urinary protein/creatinine ration, anti-dsDNA, C3, C4 and urinary β2-microglobulin. In the present paper, we evaluated in serum: Results We detected high lipid peroxidation, elevated oxidative DNA damage, excess accumulation of reactive carbonylic compounds, important oxidation of carbohydrates, disulphide bonds formation and high nitrotyrosination with statistically significant differences between groups, when compared LN and non-LN groups with control group. When compared LN and non-LN groups, our results showed: 3-Nitrotyrosine levels, the decrease of total and native serum thiols, pentosidine levels, sRAGE level and OGG1 activity correlated with disease activity markers in both LN and non-LN groups, while AGE correlated with disease activity only in non-LN group. Conclusion The cellular response to oxidative stimuli in SLE is concreted in the amplification of oxidative degradation of lipids, proteins, nucleic acid, hydro carbonates and in alteration of endogenous strategies for suppression /modulating oxidative stress. The defective DNA repair mechanism via OGG1 and the reduced regulatory effect of sRAGE in activation AGE-RAGE axis in LN group versus non-LN could explain alteration of renal architecture and development of renal injury.

Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycete‐specific transcription factor

Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so‐far uncharacterized function. AosR mitigates phagocyte‐derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic‐function sigma factor, SigH. This leads to the specific upregulation of the CysM‐dependent non‐canonical cysteine biosynthesis pathway through an auxiliary intragenic stress‐responsive promoter, an axis critical in detoxifying host‐derived oxidative and nitrosative radicals. Failure to upregulate AosR‐dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR‐SigH pathway is critical for detoxifying host‐derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.

Plasma thiol/disulphide homeostasis changes in patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease and inflammation and oxidative stress play important roles in its pathology. Thiol/disulphide homeostasis (TDH) is a special oxidative stress biomarker that has been found to be affected in several disorders including MS. There is no study demonstrating the effects of attack status of the relapsing-remitting multiple sclerosis (RRMS) patients on TDH levels. Our aim was to determine TDH levels in three different periods of RRMS patients and healthy individuals. The study was carried out in 29 patients with RRMS without a prior attack in the last twelve months (MS Control), 21 RRMS patients having a clinical acute attack within the last week (MS relapse), 12 of 21 MS relapse patients one month after the onset of attack and following 1000mg methylprednisolone for 7days (MS Remission) and 30 age- and sex-matched healthy individuals. TDH status was determined using an automated spectrophotometric analysis method. TDHlevels in all patient groups and control subjects were compared with each other. The lowest native thiol, total thiol levels and native thiol/total thiol ratio were found in the MS relapse patients in comparison to the MS control, MS remission groups and healthy controls. In contrast, disulphide levels, disulphide/native thiol and disulphide/total thiol ratios were highest in the MS relapse group compared to the other patient groups and healthy subjects. Our findings indicate that increased oxidative stress in RRMS patients is reflected with decreased native and total thiol and increased disulphide levels. Since the formation of disulphide bonds is reversible, the progression of RRMS involving abnormal TDH may be controlled, converting disulphides to thiols. So, we suggest determining the dynamic TDH status as a novel and special biomarker in the diagnosis and prognosis of the RRMS patients.

Study of structural changes of gluten proteins during bread dough mixing by Raman spectroscopy

The aim of the present study was to evaluate Raman spectroscopy in determining changes that occur in the structure of gluten proteins induced during bread dough mixing. Raman spectra were measured directly within the dough. Three particular phases of mixing were studied: under-mixing, optimum mixing and over-mixing. A thiol blocking reagent, Tris(2-carboxyethyl)phosphine (TCEP) was then used to reduce disulphide bonds within proteins to confirm the important role of disulphide bridges in gluten network formation. For the control dough, the most important changes occurred during the optimum mixing phase when an increase in intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures was observed, combined with the hydrophobic burial of tryptophan and tyrosine residues. The addition of TCEP appeared to effectively reduce the formation of intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures and lead to a more disordered secondary protein structure.

Protein disulphide isomerase inhibition as a potential cancer therapeutic strategy.

The protein disulphide isomerase (PDI) gene family is a large, diverse group of enzymes recognised for their roles in disulphide bond formation within the endoplasmic reticulum (ER). PDI therefore plays an important role in ER proteostasis, however, it also shows involvement in ER stress, a characteristic recognised in multiple disease states, including cancer. While the exact mechanisms by which PDI contributes to tumorigenesis are still not fully understood, PDI exhibits clear involvement in the unfolded protein response (UPR) pathway. The UPR acts to alleviate ER stress through the activation of ER chaperones, such as PDI, which act to refold misfolded proteins, promoting cell survival. PDI also acts as an upstream regulator of the UPR pathway, through redox regulation of UPR stress receptors. This demonstrates the pro‐protective roles of PDI and highlights PDI as a potential therapeutic target for cancer treatment. Recent research has explored the use of PDI inhibitors with PACMA 31 in particular, demonstrating promising anti‐cancer effects in ovarian cancer. This review discusses the properties and functions of PDI family members and focuses on their potential as a therapeutic target for cancer treatment.

DNA-Mediated Stack Formation of Nanodiscs.

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.

Saltiness perception related to salt release of surimi emulsified sausages: modulation in texture and microstructure by polysaccharides

SummaryThe effect of κ‐carrageenan (CG), high acyl gellan (HAG), xanthan gum (XG) and locust bean gum (LBG) on the salt release and saltiness perception of surimi emulsified sausages was investigated. CG, LBG and XG significantly reduced the fracture strain to 62.62%, 58.56% and 58.23%, causing the formation of disintegrating gels. Microstructure observation showed that sausages with CG or LBG had porous structure, but the former formed an interpenetrating network and promoted hydrophobic interactions and the formation of disulphide bonds, while the latter was physically filled in the protein network. The maximum salt release was observed in the sausage with CG due to rapid salt diffusion from porous and interpenetrating network structure. Sensory analysis further proved that the saltiness perception of sausages was enhanced by CG. The present work provided an approach for salt reduction in sausages by facilitating salt release achieved by modulating the texture attributes and microstructure using polysaccharides.

Brief descriptions of the principles of prominent methods used to study the penetration of materials into human hair and a review of examples of their use.

Consumers are attracted to the latest fashion trends and different looks. This drives the search for novel hair treatments. Some chemicals present in hair treatment products can penetrate the hair shaft. These materials can either nourish or injure the hair cortex. Different techniques have been used to investigate the mechanism of molecule penetration and the conditions under which penetration occurs. This article reviews the techniques applied for this purpose. Various microscopy techniques are used to capture clear and colourful images to determine the diffusion pathways and the exact location of the molecules under study. However, the laborious sample preparation often leads to sample destruction since cross-sectioning is often required. While various other techniques have been successfully used for investigating the penetration methods, most of these require different amounts of work to be put in for sample preparation and instrumentation. Several spectroscopic techniques have been used to study the penetration of the molecules because of the high levels of accuracy and the quick response time of these techniques. Moreover, the samples are not damaged during the investigation.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

Complex formation of copper(II), nickel(II) and γ-L-glutamyl-L-cysteinyl-glycine (GSH)

The formation of Cu(II) and Ni(II) complexes of GSH in conditions of 2-fold GSH excess over Ni(II) was studied by MALDI MS and MS/MS. The instrumental data demonstrated that S and N of GSH are involved in the coordination metal spheres, while COOH did not participate to complexation. NiL2 is a square-planar Ni(II) specie able to form one electron redox couple with Ni-(III) complex. It was found to dimerize rapidly via disulphide bond formation in the presence of air.

Analysis of disulphide bond linkage between CoA and protein cysteine thiols during sporulation and in spores of Bacillus species.

ABSTRACTSpores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.

Super-Resolution Fluorescence Microscopy Reveals Clustering Behaviour of Chlamydia pneumoniae's Major Outer Membrane Protein.

Simple SummaryChlamydia is an infamous sexually transmitted bacterium that also has a less well-known role in human respiratory infections, which has evolved a unique cell structure to enable its survival within the body. Covering the surface of this infectious cell is a strong mesh-like network made up of many different proteins which protects the cell against damage. This research focussed on the most abundant protein within this mesh, the Major Outer Membrane Protein (MOMP), and introduced a series of mutations designed to prevent the mesh from forming completely. The effect of the mutations was visualised by adding a bright fluorescent dye to each MOMP, which was then examined with a high-resolution fluorescence microscope capable of showing us each individual cell and the MOMPs at their surface. With statistical analysis, we observed that certain mutations disrupted the connections between MOMPs, giving us greater insight into how Chlamydia forms these interactions. Chlamydia is an extremely prevalent disease amongst the global population, and whilst treatable, there is currently no available vaccine. By researching Chlamydia’s biology and its method of evading our immune system, we can not only further our understanding of this complex bacterium, but also develop novel therapeutics for its treatment and prevention.Chlamydia pneumoniae is a Gram-negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of Chlamydia is a conserved immunologically dominant protein located in the outer membrane, which, together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. MOMP has a major role in the chlamydial outer membrane complex through the formation of intermolecular disulphide bonds, although the exact interactions formed are currently unknown. Here, it is proposed that due to the large number of cysteines available for disulphide bonding, interactions occur between cysteine-rich pockets as opposed to individual residues. Such pockets were identified using a MOMP homology model with a supporting low-resolution (~4 Å) crystal structure. The localisation of MOMP in the E. coli membrane was assessed using direct stochastic optical reconstruction microscopy (dSTORM), which showed a decrease in membrane clustering with cysteine-rich regions containing two mutations. These results indicate that disulphide bond formation was not disrupted by single mutants located in the cysteine-dense regions and was instead compensated by neighbouring cysteines within the pocket in support of this cysteine-rich pocket hypothesis.

Thiol-disulphide homeostasis in patients with surgical site infections

Objectives: Introduction: In infectious diseases, various inflammatory cells are active and reactive oxygen species are produced to fight against intra cellular and extra cellular microorganisms. This leads to an increase in potential antioxidant capacity and free radical production. Thiol is an important antioxidant. Thiols enter oxidation reactions with oxidative molecules and it form disulphide bonds. The aim of this study was to evaluate the dynamic thiol/disulphide homeostasis in patients with surgical site infection (SSI). Methods: Sixty-eight patients with SSI and 66 healthy persons (control group) were included in this study. Complete blood count, biochemistry, erythrocyte sedimentation rate, C-reactive protein values and thiol levels were studied in blood of the patients at 0th day and 10th day. The blood thiol disulphide homeostasis was analysed using a new automated method developed by Erel and Neselioglu. Results: Native thiol, total thiol, albumin levels and native thiol/total thiol ratio were found significantly lower in the blood samples at day 0th compared to 10th day. There was a statistically significant difference between the patient group and the control group in IMA, native thiol, total thiol, albumin, disulphide levels, disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios Conclusions: The thiol-disulphide balance is impaired in SSI. The elevated disulphide/total thiol ratio and disulphide/native thiol ratio, and reduced native thiol/total thiol ratio indicate increased oxidation in SSI. There is also a strong association between CRP, ESH and thiol-disulphide parameters. Thiol-disulphide homeostasis may potentially be of benefit in inflammatory response. J Microbiol Infect Dis 2020; 10(3):160-166.

A novel soybean protein disulphide isomerase family protein possesses dithiol oxidation activity: identification and characterization of GmPDIL6.

Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulphide bonds, viz. oxidative folding, catalysed by the protein disulphide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active centre (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulphide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was up-regulated under ER stress. GmPDIL6 may play a role in the formation of disulphide bonds in nascent proteins for oxidative folding in the ER.