Current Protein and Peptide Science - Volume 15, Issue 5, 2014

Incidence of fungal infections has increased alarmingly in past few decades. Of the fungal pathogens, the Aspergillus fumigatus has been a major cause of allergic bronchopulmonary aspergillosis (ABPA) which has five main stages - the acute, remission, exacerbation, glucocorticoid dependent and fibrotic stage. The diagnosis of ABPA remains difficult due to its overlapping clinical and radiological features with tuberculosis and cystic fibrosis. From past few decades, the crude fractions of A. fumigatus have been used for immunodiagnosis of ABPA. Most of the detection kits based on crude fractions of A. fumigatus are quite sensitive but have low specificity. Till date 21 known and 25 predicted allergens of A. fumigatus have been identified. Of these allergens, only five recombinants (rAsp f1-f4 and f6) are commercially used for diagnosis of allergic aspergillosis. Remaining allergens of A. fumigatus have been restricted for use in specific diagnosis of ABPA, due to sharing of common antigenic epitopes with other allergens. Complete sequencing of A. fumigatus genome identified 9926 genes and the reports on the proteome of A. fumigatus have shown the presence of large number of their corresponding proteins in the pathogen. The analysis of immunoproteomes developed from crude fractions of A. fumigatus by IgG/IgE reactivity with ABPA patients and animal sera have identified the panel of new antigens. A brief description on the current status of A. fumigatus antigens is provided in this review. The implementation of advance recombinant expression and peptidomic approaches on the A. fumigatus antigens may help in the selection of appropriate molecules for the development of tools for more specific early diagnosis of ABPA, and desensitization therapies for patients of allergic disorders.

Although innate immunity primarily combats systemic infections of opportunistic fungi such as Aspergillus and Candida spp., acquired and protective immunoreactions were observed long ago in animal trials following sublethal systemic infections caused by viable fungi or after challenging animals with inactivated fungal cells. Based on these observations, fungal antigens should exist which mediate such protective immunoreactions and have in part already been identified. In this context, this review focuses primarily on the various approaches that have been used to identify protectionmediating Aspergillus-antigens and their rationale. Emphasis is placed on screening methods that have exploited genetic or proteomic approaches on the basis of the corresponding fungal genome projects. Thereby, a survey and description is given of the antigens so far known to be capable of inducing immune responses that protect animals against acquiring lethal systemic aspergillosis.

Diagnosis of immunoallergenic pathologies due to microorganisms such as hypersensitivity pneumonitis includes detection of circulating specific antibodies. Detection of precipitins has classically been performed using immunoprecipitation techniques with crude antigenic extracts from microorganisms implicated as etiologic agents. However, these techniques lack standardization because of the different composition of fungal antigenic extracts from one batch to another. Therefore, there is high interest in developing standardized serological diagnostic methods using recombinant antigens. Immunoproteomics have proved to be useful for identifying the immunogenic proteins in several microorganisms linked to hypersensitivity pneumonitis. With this approach, the causative microorganisms are first isolated from the environment of patients. Then the proteins are separated by two-dimensional electrophoresis and revealed by Western blotting with sera of different patients suffering from the disease compared to sera of asymptomatic exposed controls. Immunoreactive proteins are identified by mass spectrometry. Identified immunoreactive proteins found to be specific markers for the disease could be subsequently produced as recombinant antigens using various expression systems to develop ELISA tests. Using recombinant antigens, standardized ELISA techniques can be developed, with sensitivity and specificity reaching 80% and 90%, respectively, and more if using a combination of several antigens. Immunoproteomics can be applied to any environmental microorganisms, with the aim of proposing panels of recombinant antigens able to improve the sensitivity and standardization of serologic diagnosis of hypersensitivity pneumonitis, but also other mold-induced allergic diseases such as allergic broncho pulmonary aspergillosis or asthma.

The most prevalent skin infections are mainly caused by species of dermatophytes of the genera Trichophyton, Microsporum, and Epidermophyton that infect keratinized tissues and stratum corneum of skin and hair. Besides proteases with putative role of kinases and other enzymes, immune modulators are abundantly secreted during infection as well. The molecular mechanism used by the dermatophytes to infect and counteract the host immune response is not well understood. The defense against infections basically depends on the host's immune responses to metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. The two aspects of the immune system, the immediate hypersensitivity and delayed-type hypersensitivity against dermatophytes may be crucial to the progression and severity of skin infection. Management of the infection through species identification and molecular diagnostic techniques as well as use of novel targeted drugs in addition to conventional anti-fungal compounds is of great importance in dealing with disease onsets and outbreaks. Here we reviewed the fungal skin infections elucidating their biologic and immunologic characteristics. Reaction to fungal invasion by the infected epithelial tissue on the host side is also discussed. Moreover, determinants of protective immunity and treatment options are focused that could confer long-lasting resistance to infection.

Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications.

Solvent accessible surface area (SASA) of proteins has always been considered as a decisive factor in protein folding and stability studies. It is defined as the surface characterized around a protein by a hypothetical centre of a solvent sphere with the van der Waals contact surface of the molecule. Based on SASA values, amino acid residues of a protein can be classified as buried or exposed. There are various types of SASAs starting from relative solvent accessibility to absolute surface areas. Direct estimation of accurate SASAs of folded proteins experimentally at the atomic level is not possible. However, the SASA of a native protein can be estimated computationally from the atomic coordinates. Similarly, various simulation methods are available to compute the SASA of a protein in its unfolded state. In efforts to estimate the changes in SASA related to the protein folding, a number of the unfolded state models have been proposed. In this review, we have summarized different algorithms and computational tools for SASA estimations. Furthermore, online resources for SASA calculations and representations have also been discussed in detail. This review will be useful for protein chemists and biologists for the accurate measurements of SASA and its subsequent applications for the calculation of various biophysical and thermodynamic properties of proteins.

Protein misfolding and aggregation into amyloid structures is linked with an increasing number of nonneuropathic (either localized or systemic) and neurodegenerative human disorders. In the present review, we compile and describe methods, which have been developed to predict, detect and characterize amyloid and amyloid-like protein deposits. We focus in the state-of-the-art methodologies to study and image amyloid aggregation in-vitro, from qualitative and low-resolution techniques to methods addressed to resolve protein structures at atomic level. We also recapitulate the most relevant literature describing approaches that have been demonstrated to be able to detect and characterize protein aggregation in cells and living organisms, as well as methodologies to report cytotoxicity associated to amyloid formation. Overall, the aim of this review is to illustrate computational and experimental methods to characterize and predict in-vitro and in-vivo amyloid aggregation, providing the readers valuable information to elect the most appropriate techniques at their convenience.

The clostridial neurotoxins (CNTs) are among the most potent protein toxins known to humans. CNTs include seven serotypes (A~G) of botulinum toxins (BoNTs), which cause botulism, a flaccid paralysis, and tetanus toxin (TeNT), which causes spastic paralysis. BoNTs are classified as category A agent and may be used as potential bioterrorism weapons. On the other hand, the ability of an extremely low dosage of BoNTs (less than 1 ng) to cause reversible partial paralysis upon injection into muscle has turned BoNTs, in particular serotypes A and B, from deadly agents to novel therapeutic agents for treatment of a wide range of clinical conditions associated with involuntary muscle spasm and contractions. In addition to clinical use, they may also be used in cosmetics. Further indications for BoNTs will continue to be developed, although current BoNT therapies have encountered some limitations due to the pharmacological properties of BoNTs, such as their ability to elicit immunoresistance in patients upon periodical injections. This review summarizes the present knowledge of the mechanisms of action of CNTs, with particular focus on the mode of substrate recognition by CNT catalytic domains and knowledge regarding substrate recognition can be utilized to develop novel BoNT products to extend its usefulness in therapeutic interventions and overcome the immunoresistance problems.

MPN (Mpr1/Pad1 N-terminal) domain-containing proteins are present throughout all domains of life. In eukaryotes, MPN domain-containing proteins are commonly found in association with other molecules in large protein complexes, where examples comprise; the 26S proteasome and the COP9 (Constitutive photomorphogenesis 9) signalosome complexes, including the MPN subunits, POH1 and Mov34, CSN5 and CSN6, respectively. Examples of MPN domaincontaining proteins that are not incorporated in a large multi-protein complex have also been reported and include AMSH (for associated molecule with the SH3 domain of STAM) and the AMSH-Like Protein (AMSH-LP). Within the MPN domain super-family, two main subclasses have been characterised: the MPN+ and MPN- domain-containing proteins. MPN+ domain-containing proteins are classified as metalloenzymes responsible for isopeptidase activity. These proteins display a JAMM (JAB1-MPN-MOV34) metalloisopeptidase motif, typically consisting of a canonical sequence (E-x[2]-H-S/T-Hx[ 7]-S-x[2]-D) and coordinating a zinc ion. The JAMM motif specifies a catalytic centre essential for selective hydrolysis of linkages, contained between ubiquitin/ubiquitin-like proteins and target proteins or between ubiquitin monomers within a polymeric chain. The MPN- family classifies proteins, which lack the key residues present in the typical JAMM motif. These MPN- proteins are void of catalytic activity, but recent studies have proposed a role in mediating protein-protein interactions, in acting as a scaffold or in activity regulation. In light of recent structural and functional studies, a more detailed understanding of these proteins has been gained and is given in the present review.