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Ankylosing Spondylitis is an autoimmune disease leading to inflammation in the joints and ligaments of the spine. ERAP1 is a major risk factor for AS and ERAP1 mutations may result in structural changes that alter the trimming efficiency, thereby altering the immune response. The underlying structural mechanisms of AS pathogenesis have not yet been fully elucidated. This study investigated ERAP1/ERAP2 allotypes using Molecular Dynamics in both monomeric and dimeric forms. ERAP1's domain IV has been found to be a favorable region for dimerization. Different allotype dimers exhibited different stability characteristics. Furthermore, the effects of allotypic variation were more pronounced in Hap2-/Hap8-coupled dimer structures and were more distinct in heterodimers. An analysis of the interchain region revealed that both H-bonding and electrostatic interactions between chains of Hap2–N392 heterodimer structures were lower than those between Hap2–Hap2 revealing that allotypic variations played a significant role in stabilizing and destabilizing dimer structures.

Summary The secretomes of filamentous fungi contain a diversity of small secreted cysteine‐rich proteins (SSCPs) that have a variety of properties ranging from toxicity to surface activity. Some SSCPs are recognized by other organisms as indicators of fungal presence, but their function in fungi is not fully understood. We detected a new family of fungal surface‐active SSCPs (saSSCPs), here named hyphosphere proteins (HFSs). An evolutionary analysis of the HFSs in Pezizomycotina revealed a unique pattern of eight single cysteine residues (C‐CXXXC‐C‐C‐C‐C‐C) and a long evolutionary history of multiple gene duplications and ancient interfungal lateral gene transfers, suggesting their functional significance for fungi with different lifestyles. Interestingly, recombinantly produced saSSCPs from three families (HFSs, hydrophobins and cerato‐platanins) showed convergent surface‐modulating activity on glass and on poly(ethylene‐terephthalate), transforming their surfaces to a moderately hydrophilic state, which significantly favoured subsequent hyphal attachment. The addition of purified saSSCPs to the tomato rhizosphere had mixed effects on hyphal attachment to roots, while all tested saSSCPs had an adverse effect on plant growth in vitro. We propose that the exceptionally high diversity of saSSCPs in Trichoderma and other fungi evolved to efficiently condition various surfaces in the hyphosphere to a fungal‐beneficial state.

Background: Senescent cells accumulate in tissues over time as part of the natural aging process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to eliminate senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modeling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cancer cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it co-localizes with FOXO4 foci and binds FOXO4 to a greater extent compared to TP53. Finally, we show that concomitant intra-tumoral injection of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis in a transgenic mouse model of melanoma. Interpretation: Taken together, our results reveal that peptides can be generated to specifically eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding Statement: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University. Declaration of Interests: S.S. Cinaroglu, Y. Ahiska, U. Sezerman, G.B. Akcapinar, E. Timucin have a patent for the ES2 structure and are members of a company, Eternans Ltd. that is working on ES2 for therapeutic use. No potential conflicts of interest were disclosed by the other authors. Ethics Approval Statement: All experiments were approved by Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee (IACUC) (TR01_IP00000674) and conformed to the guidelines set by United States Animal Welfare Act and the National Institutes of Health.

Fungal evolutionary biology is impeded by the scarcity of fossils, irregular life cycles, immortality, and frequent asexual reproduction. Simple and diminutive bodies of fungi develop inside a substrate and have exceptional metabolic and ecological plasticity, which hinders species delimitation. However, the unique fungal traits can shed light on evolutionary forces that shape the environmental adaptations of these taxa. Higher filamentous fungi that disperse through aerial spores produce amphiphilic and highly surface-active proteins called hydrophobins (HFBs), which coat spores and mediate environmental interactions. We exploited a library of HFB-deficient mutants for two cryptic species of mycoparasitic and saprotrophic fungi from the genus Trichoderma (Hypocreales) and estimated fungal development, reproductive potential, and stress resistance. HFB4 and HFB10 were found to be relevant for Trichoderma fitness because they could impact the spore-mediated dispersal processes and control other fitness traits. An analysis in silico revealed purifying selection for all cases except for HFB4 from T. harzianum, which evolved under strong positive selection pressure. Interestingly, the deletion of the hfb4 gene in T. harzianum considerably increased its fitness-related traits. Conversely, the deletion of hfb4 in T. guizhouense led to the characteristic phenotypes associated with relatively low fitness. The net contribution of the hfb4 gene to fitness was found to result from evolutionary tradeoffs between individual traits. Our analysis of HFB-dependent fitness traits has provided an evolutionary snapshot of the selective pressures and speciation process in closely related fungal species.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness., Author summary Hydrophobins (HFBs) are unique, non-toxic small fungal proteins with outstandingly high surface activity. They modulate surface properties by self-assembling at interfaces and have a multitude of applications for drug delivery, biosensors, and cosmetics. To explore the applied potential of HFBs, we investigated their functions in the naturally HFB-enriched Trichoderma spp. fungi (Ascomycota). We show that HFBs unexpectedly specifically accumulate inside aerial hyphae, where they associate with lipid-enriched organelles and putatively line up the vacuolar structures, which contribute to the structure and longevity of aerial mycelium. The maximum of HFB synthesis and secretion happens before the sporulation, providing a massive extracellular matrix suitable for the protective coating of rapidly produced spores. Furthermore, HFBs are involved in the water sensing mechanism of spores and thus are linked to the dormancy/germination switch. Our results reveal that HFBs have a broad range of intracellular functions and are essential for fungal development and fitness.

Background: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. Interpretation: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health \& Science University. (C) 2021 The Authors. Published by Elsevier B.V.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity.

Trichoderma guizhouense NJAU 4742 (Harzianum clade) can suppress the causative agent of banana wild disease Fusarium oxysporum f. sp. cubense 4 (Foc4). To identify genes involved in this trait, we used T-DNA insertional mutagenesis and isolated one mutant that was unable to overgrow Foc4 and had reduced antifungal ability. Using the high-efficiency thermal asymmetric interlaced-PCR, the T-DNA was located in the terminator of a neutral metalloprotease gene (encoding a MEROPS family M35 protease), which was named nmp1. The antifungal activity of the mutant was recovered by retransformation with wild-type nmp1 gene. The purified NMP1 (overexpressed in Pichia pastoris) did not inhibit the growth and germination of other fungi in vitro. Its addition, however, partly recovered the antifungal activity of the mutant strain against some fungi. The expression of nmp1 is induced by the presence of fungi and by dead fungal biomass, but the time-course of transcript accumulation following the physical contact depends on mode of interaction: it increases in cases of long-lasting parasitism and decreases if the prey fungus is dead shortly after or even before the contact (predation). We thus conclude that NMP1 protein of T. guizhouense has major importance for mycotrophic interactions and defence against other fungi.

Hydrophobins (HFBs) are small surface-active proteins secreted by filamentous fungi. Being amphiphilic, they spontaneously form layers that convert surfaces from hydrophilic to hydrophobic and vice versa. We have compared properties of the class II HFB4 and HFB7 from Trichoderma virens as produced in Escherichia coli and Pichiapastoris. Since the production in E. coli required denaturation/renaturation steps because of inclusion bodies, this treatment was also applied to HFBs produced and secreted in yeast. The protein yields for both systems were similar. Both HFBs produced by E. coli proved less active on PET compared to HFBs produced in P. pastoris. HFBs produced in E. coli decreased the hydrophilicity of glass the most, which correlated with the adsorption of a more dense protein layer on glass compared to HFBs produced in P. pastoris. The hydrophobins produced in P. pastoris formed highly structured monolayers. Layers of hydrophobins produced in E. coli were less prone to self-organization. Our data suggests that irrespective of the production host, the HFBs could be used in various applications that are based on their surface activity. However, the production host and the subsequent purification procedure will influence the stability of HFB layers. In the area of high-value biomedical devices and nanomaterials, where the formation of highly ordered protein monolayers is essential, our results point to P. pastoris as the preferred production host. Furthermore, the choice of an appropriate hydrophobin for a given application appears to be equally important. (C) 2017 The Authors. Published by Elsevier B.V.

Metal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation of metal ions or clusters in proteins. Approximately one-third of these proteins in the databases contain metal ions. Many biological and chemical processes in nature involve metal ion-binding proteins, aka metalloproteins. Many cellular reactions that underpin life require metalloproteins. Most of the remarkable, complex chemical transformations are catalysed by metalloenzymes. Realization of the importance of metal-binding sites in a variety of cellular events led to the advancement of various computational methods for their prediction and characterization. Furthermore, as structural and functional knowledgebase about metalloproteins is expanding with advances in computational and experimental fields, the focus of the research is now shifting towards de novo design and redesign of metalloproteins to extend nature’s own diversity beyond its limits. In this review, we will focus on the computational toolbox for prediction of metal ion-binding sites, de novo metalloprotein design and redesign. We will also give examples of tailor-made artificial metalloproteins designed with the computational toolbox.

Recent advancements in omics technologies are instrumental in studying to molecular events and to reveal the underlying mechanisms. In this chapter we focus on use of different types of omics data to understand the fungal gene expression and translation mechanism. These data were analyzed in a system biology approach providing valuable insights for efficient use of fungal expression systems to achieve high production yields.

In this study, a major cellulase, namely endoglucanase 1 (EGI) from Trichoderma reesei was mutated by the introduction of four different lysine and glycine rich loops to create a hotspot for directed crosslinking of EGI away from the active site. The impact of the inserted loops on the stability of the enzyme was analyzed using molecular dynamics (MD) and the effect on the active site was studied using molecular mechanics (MM) simulations. The best loop mutation predicted in silico (EGI_L5) was introduced to EGI via site directed mutagenesis. The loop mutant EGI_L5 and EGI were both expressed in Pichia pastoris. Enzymes were characterized and their activities against soluble substrates such as CMC and 4-MUC were determined. Both enzymes exhibited similar pH and temperature activity and thermal stability profiles. Moreover, specific activity of EGI_L5 against 4-MUC was found to be the same as the native enzyme. (C) 2012 Elsevier B.V. All rights reserved.

Trichoderma guizhouense NJAU 4742 (Harzianum clade) can suppress the causative agent of banana wild disease Fusarium oxysporum f. sp. cubense 4 (Foc4). To identify genes involved in this trait, we used T-DNA insertional mutagenesis and isolated one mutant that was unable to overgrow Foc4 and had reduced antifungal ability. Using the high-efficiency thermal asymmetric interlaced-PCR, the T-DNA was located in the terminator of a neutral metalloprotease gene (encoding a MEROPS family M35 protease), which was named nmp1. The antifungal activity of the mutant was recovered by retransformation with wild-type nmp1 gene. The purified NMP1 (overexpressed in Pichia pastoris) did not inhibit the growth and germination of other fungi in vitro. Its addition, however, partly recovered the antifungal activity of the mutant strain against some fungi. The expression of nmp1 is induced by the presence of fungi and by dead fungal biomass, but the time-course of transcript accumulation following the physical contact depends on mode of interaction: it increases in cases of long-lasting parasitism and decreases if the prey fungus is dead shortly after or even before the contact (predation). We thus conclude that NMP1 protein of T. guizhouense has major importance for mycotrophic interactions and defence against other fungi.

In the last decades, effective cellulose degradation became a major point of interest due to the properties of cellulose as a renewable energy source and the widespread application of cellulases (the cellulose degrading enzymes) in many industrial processes. Effective bioconversion of lignocellulosic biomass into soluble sugars for ethanol production requires use of thermostable and highly active cellulases. The library of current cellulases includes enzymes that can work at acidic and neutral pH in a wide temperature range. However, only few cellulases are reported to be thermostable. In order to alleviate this, we have performed a hybrid approach for the thermostabilization of a key cellulase, Endoglucanase I (EGI) from Trichoderma reesei. We combined in silico and in vitro experiments to modulate the thermostability of EGI. Four different predictive algorithms were used to set up a library of mutations. Three thermostabilizer mutations (Q126F, K272F, Q274V) were selected and molecular dynamics simulations at room temperature and high temperatures were performed to analyze the effect of the mutations on enzyme structure and stability. The mutations were then introduced into the endoglucanase 1 gene, using site-directed mutagenesis, and the effect of the mutations on enzyme structure and stability were determined. MD simulations supported the fact that Q126F, K272F and Q274V mutations have a thermostabilizing effect on the protein structure. Experimental studies validated that all of the mutants exhibited higher thermostability compared with native EGI albeit with a decrease in specific activity.

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

Trichoderma reesei cellulases are important biocatalysts for a wide range of industrial applications that include the paper, feed, and textile industries. T. reesei endoglucanase 1 (egl1) was successfully expressed as an active and stable catalyst in Pichia pastoris for the first time. Codon optimization was applied to egl1 of T. reesei to enhance its expression levels in P. pastoris. When compared with the originally cloned egl1 gene of T. reesei, the synthetic codon optimized egl1 gene (egl1s) was expressed at a higher level in P. pastoris. Batch fermentations of both clones with the same copy number under controlled conditions indicated that codon optimized EGI enzyme activity increased to 1.24 fold after 72 h of methanol induction. Our research indicated that P. pastoris is a suitable host for cellulase production. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 12571263, 2011