The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

Application of MALDI-TOF analysis to reveal diversity and dynamics of winemaking yeast species in wild-fermented, organically produced, New Zealand Pinot Noir wine.

Rapid yeast identification is of particular importance in monitoring wine fermentation and assessing strain application in winemaking. We used MALDI-TOF MS analysis supported by 26 S rRNA gene sequence analysis and Saccharomyces-specific PCR testing to differentiate reference and field strains recovered from organic wine production facilities in Waipara, New Zealand, in which Pinot Noir wine was produced by spontaneous fermentations in the vineyard and in the winery. Strains were isolated from each of four key stages of each ferment to evaluate changes in taxonomic diversity. MALDI-TOF MS analysis was confirmed as an excellent yeast identification method, with even closely related Saccharomyces species readily distinguished. A total of 13 indigenous species belonging to eight genera were identified from Pinot Noir ferments, with taxonomic diversity generally reducing as fermentation progressed. However, differences between the taxa recovered were observed between the vineyard and winery ferments, despite the grapes used being from the same batch. Furthermore, some consistent proteomic differences between strains of S. cerevisiae, Hanseniasporum uvarum, Candida californica, Pichia membranifaciens and Starmerella bacillaris correlated with the different fermentation systems used. The high speed, low cost, taxonomic resolution and ability to characterise subtle changes in phenotype that may result from variations in environmental conditions makes MALDI-TOF analysis an attractive tool for further and wider applications in the wine industry. Such applications may include monitoring wine fermentation to actively support the consistency of high-quality wine products, and potentially for the development of such products too.

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents.

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 µM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC50 = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC50/EC50) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 µM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.

A Multi-Bioassay Integrated Approach to Assess the Antifouling Potential of the Cyanobacterial Metabolites Portoamides.

The cyclic peptides portoamides produced by the cyanobacterium Phormidium sp. LEGE 05292 were previously isolated and their ability to condition microcommunities by allelopathic effect was described. These interesting bioactive properties are, however, still underexplored as their biotechnological applications may be vast. This study aims to investigate the antifouling potential of portoamides, given that a challenge in the search for new environmentally friendly antifouling products is to find non-toxic natural alternatives with the ability to prevent colonization of different biofouling species, from bacteria to macroinvertebrates. A multi-bioassay approach was applied to assess portoamides antifouling properties, marine ecotoxicity and molecular mode of action. Results showed high effectiveness in the prevention of mussel larvae settlement (EC50 = 3.16 µM), and also bioactivity towards growth and biofilm disruption of marine biofouling bacterial strains, while not showing toxicity towards both target and non-target species. Antifouling molecular targets in mussel larvae include energy metabolism modifications (failure in proton-transporting ATPases activity), structural alterations of the gills and protein and gene regulatory mechanisms. Overall, portoamides reveal a broad-spectrum bioactivity towards diverse biofouling species, including a non-toxic and reversible effect towards mussel larvae, showing potential to be incorporated as an active ingredient in antifouling coatings.

Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization.

Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs. As a preliminary step towards their biophysical and structural characterization, we have expressed and purified a HS KAP (KAP11.1) and a HGT KAP (KAP6.1). The expression and purification of KAPs is challenging because they are cysteine-rich proteins with unusual amino acid compositions, they tend to be insoluble in isolation and are prone to forming aggregates in solution. Here we describe the high yield production of pure, soluble KAPs in a chaotrope- and detergent-free buffer. This method has the potential to be used for the overproduction of other KAPs and similar cysteine-rich proteins with high isoelectric points.

Diversity of Trichocyte Keratins and Keratin Associated Proteins.

Wool and hair fibres are primarily composed of proteins of which the keratins and keratin associated proteins (KAPs) are the major component. Considerable diversity is known to exist within these two groups of proteins. In the case of the keratins two major families are known, of which there are 11 members in the acidic Type I family and 7 members in the neutral-basic Type II family. The KAPs are even more diverse than the keratins, with 35 families being known to exist when the KAPs found in monotremes, marsupials and other mammalian species are taken into consideration. Human hair and wool are known to have 88 and 73 KAPs respectively, though this number rises for wool when polymorphism within KAP families is included.

Fibre Ultrastructure.

Mammalian hair fibres can be structurally divided into three main components: a cuticle, cortex and sometimes a medulla. The cuticle consists of a thin layer of overlapping cells on the surface of the fibre, constituting around 10% of the total fibre weight. The cortex makes up the remaining 86-90% and is made up of axially aligned spindle-shaped cells of which three major types have been recognised in wool: ortho, meso and para. Cortical cells are packed full of macrofibril bundles, which are a composite of aligned intermediate filaments embedded in an amorphous matrix. The spacing and three-dimensional arrangement of the intermediate filaments vary with cell type. The medulla consists of a continuous or discontinuous column of horizontal spaces in the centre of the cortex that becomes more prevalent as the fibre diameter increases.

The Follicle Cycle in Brief.

This chapter presents a very succinct overview of the cyclic biology of the hair follicle as it transitions from the quiescent telogen stage to the anagen stage in which hairs are actively produced before regressing through the catagen stage to telogen.

Development of Hair Fibres.

The growth of hairs occurs during the anagen phase of the follicle cycle. Hair growth begins with basement membrane-bound stem cells (mother cells) around the dermal papilla neck which continuously bud off daughter cells which further divide as a transient amplifying population. Division ceases as cell line differentiation begins, which entails changes in cell junctions, cell shape and position, and cell-line specific cytoplasmic expression of keratin and trichohyalin. As the differentiating cells migrate up the bulb, nuclear function ceases in cortex, cuticle and inner root sheath (IRS) layers. Past the top of the bulb, cell shape/position changes cease, and there is a period of keratin and keratin-associated protein (KAP) synthesis in fibre cell lines, with increases, in particular of KAP species. A gradual keratinization process begins in the cortex at this point and then non-keratin cell components are increasingly broken down. Terminal cornification, or hardening, is associated with water loss and precipitation of keratin. In the upper follicle, the hair, now in its mature form, detaches from the IRS, which is then extracted of material and becomes fragmented to release the fibre. Finally, the sebaceous and sudoriferous (if present) glands coat the fibre in lipid-rich material and the fibre emerges from the skin. This chapter follows the origin of the hair growth in the lower bulb and traces the development of the various cell lines.

Preparation of wool follicles for proteomic studies.

A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs.

The proteomics of wool fibre morphogenesis.

Gel and gel-free proteomic techniques have been used for the first time to directly study the proteins present in whole wool follicles and dissected portions of follicles that correlated with morphological changes in the developing fibre as determined by transmission electron microscopy. Individual wool follicles were dissected into four portions designated as the bulb, elongation, keratogenous and keratinisation portions. Gel-free proteomic analysis of dissected portions from 30 follicles showed that the first keratins to appear were K31, K35 and K85, in the bulb portion. The first epithelial KAP, trichohyalin, was detected in the bulb portion and the first cortical KAP, KAP11.1 was found in the elongation portion. Other major trichocyte keratins and cortical KAPs began to appear further up the follicle in the keratogenous and keratinisation zones. These results were consistent with what has been observed from gene expression studies and correlated well with the morphological changes observed in the follicle. Other proteins detected by this approach included the keratin anchor protein desmoplakin, as well as vimentin and epithelial keratins, histones, ribosomal proteins and collagens. Two-dimensional electrophoretic (2DE) analysis of dissected portions of 50 follicles revealed substantial changes in the position, number and intensity of the spots of the trichocyte keratins as they progressed through the follicle zones, suggesting that they are subject to modification as a result of the keratinisation process. Also present in the 2DE maps were a number of epithelial keratins, presumably from the inner and outer root sheaths, and the dermal components.

Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex.

The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained.

Modeling deamidation in sheep α-keratin peptides and application to archeological wool textiles.

Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wool's α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.

Protein oxidation: identification and utilisation of molecular markers to differentiate singlet oxygen and hydroxyl radical-mediated oxidative pathways.

The effect of reactive oxidation species (ROS) on tryptophan or tyrosine was investigated by qualitatively determining the major detectable oxidation products generated by hydroxyl radicals, produced by the Fenton process, or singlet oxygen, generated by exposure to green light in the presence of Rose Bengal, on these photosensitive amino acids in synthetic pentapeptides. Based on mass spectrometric analysis it would appear that the hydroxyl radical favours a pathway leading to the formation of tryptophandione-based products from tryptophan. In contrast singlet oxygen attack appears to favour the formation of kynurenine-type products from tryptophan. Specific oxidative products observed proteomically are therefore potentially able to discriminate between predominant ROS-mediated pathways. To validate these findings, a keratin-enriched extract was exposed to UVB light under aqueous conditions. The observation of the conversion of tryptophan to hydroxytryptophan in marker peptides, and the absence of singlet-oxygen specific modifications, suggested that under these conditions oxidative degradation occurred primarily via hydroxyl radical attack. These observations provide the first direct proteomic evidence of the dominant photodegradation pathways in wet wool.

Predictive Potential of MALDI-TOF Analyses for Wine and Brewing Yeast.

The potential of MALDI-TOF profiling for predicting potential applications of yeast strains in the beverage sector was assessed. A panel of 59 commercial yeasts (47 wine and 12 brewing yeasts) was used to validate the concept whereby 2 culture media (YPD agar and YPD broth), as well as two mass ranges m/z 500-4000 and m/z 2000-20,000, were evaluated for the best fit. Three machine learning-based algorithms, PCA, MDS, and UMAP, in addition to a hierarchical clustering method, were employed. Profiles derived from broth cultures yielded more peaks, but these were less well-defined compared with those from agar cultures. Hierarchical clustering more clearly resolved different species and gave a broad overview of potential strain utility, but more nuanced insights were provided by MDS and UMAP analyses. PCA-based displays were less informative. The potential of MALDI-TOF proteomics in predicting the utility of yeast strains of commercial benefit is supported in this study, provided appropriate approaches are used for data generation and analysis.

Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2).

Keratin-associated proteins (KAPs) are a major component of wool and other keratin-containing tissues. While four KAP1-n proteins have been identified in sheep, only three genes have been described encoding KAP1-1, KAP1-3 and KAP1-4. Here, we used a sequence conserved across the known KAP1-n genes to search the inaugural Ovine Genome Sequence (v1.0) and identified a new KAP1-n sequence on chromosome 11. PCR amplification of this sequence revealed an open reading frame of 474-bp that putatively encodes a polypeptide sequence very similar to the previously described ovine KAP1-2 protein and suggests that the newly identified sequence represents the previously unidentified KAP1-2 gene (KRTAP1-2). Its expression in skin was confirmed by PCR, and the mRNA was localized to the cortex of the mid-keratinization zone of a growing wool fibre using a gene-specific probe and in situ hybridization. PCR-SSCP analysis of KRTAP1-2 revealed nine unique banding patterns representing nine different DNA sequences. One sequence was identical to, and the other eight were homologous to, the sequence identified above, suggesting that they were allelic variants of ovine KRTAP1-2. There were ten single nucleotide substitutions identified, although only three of these were non-synonymous and would potentially result in amino acid changes. The variation identified here may influence the expression or protein structure of KAP1-2 and consequently wool structure and wool traits.

Combination of acid labile detergent and C18 Empore™ disks for improved identification and sequence coverage of in-gel digested proteins.

A protocol for improved extraction of peptides from in-gel protein digests, using a combination of the acid labile surfactant, sodium deoxycholate (SDC) and C18 Empore™ membranes, is presented. This approach results in better mass spectrum quality, higher numbers of identified peptide peaks and improved identification scores compared to standard tryptic digestion protocols, or protocols using only SDC or only C18 Empore™ disks. The advantages of the new protocol are demonstrated for two different types of samples: Merino wool intermediate filament proteins and Elaeis guineensis (oil palm) mesocarp proteins.

The influence of growth conditions on MALDI-TOF MS spectra of winemaking yeast: implications for industry applications.

Previous studies have shown MALDI-TOF MS to be a powerful tool in wine yeast identification and potential prediction of application. However, it is also established that substrate composition influences protein expression, but the degree to which this may affect MALDI-TOF spectra (and analytical results thereof) has not been fully explored. To further inform assay optimisation, the influence on MALDI-TOF spectra was determined using eight Saccharomyces strains of diverse origins cultivated on grape juices from Pinot Noir and Chardonnay varieties, synthetic grape juice, and laboratory-grade artificial culture media (YPD broth and agar). Our results demonstrated significant influences of culture media on strain MALDI-TOF spectra. Yeast culture on YPD agar is recommended for taxonomic studies, with YPD broth culture of S. cerevisiae offering improved intra-subspecific differentiation Furthermore, our data supported a correlation between MALDI spectra and the potential industrial application of individual yeast strains.