The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana.

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production.

SARS-CoV-2 humoral responses following booster BNT162b2 vaccination in patients with B-cell malignancies.

Patients with B-cell malignancies have suboptimal immune responses to SARS-CoV-2 vaccination and are a high-risk population for severe COVID19 disease. We evaluated the effect of a third booster BNT162b2 vaccine on the kinetics of anti- SARS-CoV-2 neutralizing antibody (NAbs) titers in patients with B-cell malignancies. Patients with NHL (n = 54) Waldenström's macroglobulinemia (n = 90) and chronic lymphocytic leukemia (n = 49) enrolled in the ongoing NCT04743388 study and compared against matched healthy controls. All patient groups had significantly lower NAbs compared to controls at all time points. 1 month post the third dose (M1P3D) NAbs increased significantly compared to previous time points (median NAbs 77.9%, p < .05 for all comparisons) in all patients. NAbs ≥ 50% were seen in 59.1% of patients, 34.5% of patients with suboptimal responses post-second dose, elicited a protective NAb titer ≥50%. Active treatment, rituximab, and BTKi treatment were the most important prognostic factors for a poor NAb response at 1MP3D; only 25.8% of patients on active treatment had NAbs ≥ 50%. No significant between-group differences were observed. Patients with B-cell malignancies have inferior humoral responses against SARS-CoV-2 and booster dose enhances the NAb response in a proportion of these patients.

Linear B-Cell Epitope Prediction for In Silico Vaccine Design: A Performance Review of Methods Available via Command-Line Interface.

Linear B-cell epitope prediction research has received a steadily growing interest ever since the first method was developed in 1981. B-cell epitope identification with the help of an accurate prediction method can lead to an overall faster and cheaper vaccine design process, a crucial necessity in the COVID-19 era. Consequently, several B-cell epitope prediction methods have been developed over the past few decades, but without significant success. In this study, we review the current performance and methodology of some of the most widely used linear B-cell epitope predictors which are available via a command-line interface, namely, BcePred, BepiPred, ABCpred, COBEpro, SVMTriP, LBtope, and LBEEP. Additionally, we attempted to remedy performance issues of the individual methods by developing a consensus classifier, which combines the separate predictions of these methods into a single output, accelerating the epitope-based vaccine design. While the method comparison was performed with some necessary caveats and individual methods might perform much better for specialized datasets, we hope that this update in performance can aid researchers towards the choice of a predictor, for the development of biomedical applications such as designed vaccines, diagnostic kits, immunotherapeutics, immunodiagnostic tests, antibody production, and disease diagnosis and therapy.

Extended Human G-Protein Coupled Receptor Network: Cell-Type-Specific Analysis of G-Protein Coupled Receptor Signaling Pathways.

G-protein coupled receptors (GPCRs) mediate crucial physiological functions in humans, have been implicated in an array of diseases, and are therefore prime drug targets. GPCRs signal via a multitude of pathways, mainly through G-proteins and ß-arrestins, to regulate effectors responsible for cellular responses. The limited number of transducers results in different GPCRs exerting control on the same pathway, while the availability of signaling proteins in a cell defines the result of GPCR activation. The aim of this study was to construct the extended human GPCR network (hGPCRnet) and examine the effect that cell-type specificity has on GPCR signaling pathways. To achieve this, protein-protein interaction data between GPCRs, G-protein coupled receptor kinases (GRKs), Gα subunits, ß-arrestins, and effectors were combined with protein expression data in cell types. This resulted in the hGPCRnet, a very large interconnected network, and similar cell-type-specific networks in which, distinct GPCR signaling pathways were formed. Finally, a user friendly web application, hGPCRnet ( http://bioinformatics.biol.uoa.gr/hGPCRnet ), was created to allow for the visualization and exploration of these networks and of GPCR signaling pathways. This work, and the resulting application, can be useful in further studies of GPCR function and pharmacology.

ANTISOMA: A Computational Pipeline for the Reduction of the Aggregation Propensity of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) constitute a promising class of therapeutics, since ca. 25% of all biotech drugs in development are mAbs. Even though their therapeutic value is now well established, human- and murine-derived mAbs do have deficiencies, such as short in vivo lifespan and low stability. However, the most difficult obstacle to overcome, toward the exploitation of mAbs for disease treatment, is the prevention of the formation of protein aggregates. ANTISOMA is a pipeline for the reduction of the aggregation tendency of mAbs through the decrease in their intrinsic aggregation propensity, based on an automated amino acid substitution approach. The method takes into consideration the special features of mAbs and aims at proposing specific point mutations that could lead to the redesign of those promising therapeutics, without affecting their epitope-binding ability. The method is available online at http://bioinformatics.biol.uoa.gr/ANTISOMA .

Analysis of Single-Nucleotide Polymorphisms in Human Voltage-Gated Ion Channels.

Voltage-gated ion channels (VGICs) are one of the largest groups of transmembrane proteins. Due to their major role in the generation and propagation of electrical signals, VGICs are considered important from a medical viewpoint, and their dysfunction is often associated with Channelopathies. We identified disease-associated mutations and polymorphisms in these proteins through mapping missense single-nucleotide polymorphisms from the UniProt and ClinVar databases on their amino acid sequence, considering their special topological and functional characteristics. Statistical analysis revealed that disease-associated SNPs are mostly found in the voltage sensor domain and the pore loop. Both of these regions are extremely important for the activation and ion conductivity of VGICs. Moreover, among the most frequently observed mutations are those of arginine to glutamine, to histidine or to cysteine, which can probably be attributed to the extremely important role of arginine residues in the regulation of membrane potential in these proteins. We suggest that topological information in combination with genetic variation data can contribute toward a better evaluation of the effect of currently unclassified mutations in VGICs. It is hoped that potential associations with certain disease phenotypes will be revealed in the future with the use of similar approaches.

Delving into the amyloidogenic core of human leukocyte chemotactic factor 2.

ALECT2 (leukocyte chemotactic factor 2) amyloidosis is one of the most recently identified amyloid-related diseases, with LECT2 amyloids commonly found in different types of tissues. Under physiological conditions, LECT2 is a 16 kDa multifunctional protein produced by the hepatocytes and secreted into circulation. The pathological mechanisms causing LECT2 transition into the amyloid state are still largely unknown. In the case of ALECT2 patients, there is no disease-causing mutation, yet almost all patients carry a common polymorphism that appears to be necessary but not sufficient to directly trigger amyloidogenesis. In this work, we followed a reductionist methodology in order to detect critical amyloidogenic "hot-spots" during the fibrillation of LECT2. By associating experimental and computational assays, this approach reveals the explicit amyloidogenic core of human LECT2 and pinpoints regions with distinct amyloidogenic properties. The fibrillar architecture of LECT2 polymers, based on our results, provides a wealth of detailed information about the amyloidogenic "hot-spot" interactions and represents a starting point for future peptide-driven intervention in ALECT2 amyloidosis.

The gram-negative outer membrane modeler: Automated building of lipopolysaccharide-rich bacterial outer membranes in four force fields.

Outer membranes are a crucial component of Gram-negative bacteria, containing standard lipids in their inner leaflet, lipopolysaccharides (LPSs) in their outer leaflet, and transmembrane ß-barrels known as outer membrane proteins (OMPs). OMPs regulate functions such as substrate transport and cell movement, while LPSs act as a protective barrier for bacteria and can cause toxic reactions in humans. However, the experimental study of outer membranes is challenging. Molecular dynamics simulations are often used for the computational study of membrane systems, but the preparation of complex, LPS-rich outer membranes is not straightforward. The Gram-Negative Outer Membrane Modeler (GNOMM) is an automated pipeline for preparing simulation systems of OMPs embedded in LPS-containing membranes in four different force fields. Given the physiological and clinical importance of outer membranes and their components, GNOMM can be a useful tool in the study of their structure, function, and implications in diseases. GNOMM is available at http://bioinformatics.biol.uoa.gr/GNOMM. © 2019 Wiley Periodicals, Inc.

Hidden Aggregation Hot-Spots on Human Apolipoprotein E: A Structural Study.

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aß peptide in amyloid plaques in Alzheimer's disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer's disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE-Αß complex. Our in vitro biophysical results prove that apoE peptide-analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE-Αß complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αß; taking an important step forward in the field of Alzheimer's anti-aggregation drug development.

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer.

Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. "Acetyl-chromatin" homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation.

Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients.

BACKGROUND: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. METHODS: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. RESULTS: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. CONCLUSIONS: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

αCGRP, another amyloidogenic member of the CGRP family.

The Calcitonin-gene related peptide (CGRP) family is a group of peptide hormones, which consists of IAPP, calcitonin, adrenomedullin, intermedin, αCGRP and ßCGRP. IAPP and calcitonin have been extensively associated with the formation of amyloid fibrils, causing Type 2 Diabetes and Medullary Thyroid Carcinoma, respectively. In contrast, the potential amyloidogenic properties of αCGRP still remain unexplored, although experimental trials have indicated its presence in deposits, associated with the aforementioned disorders. Therefore, in this work, we investigated the amyloidogenic profile of αCGRP, a 37-residue-long peptide hormone, utilizing both biophysical experimental techniques and Molecular Dynamics simulations. These efforts unravel a novel amyloidogenic member of the CGRP family and provide insights into the mechanism underlying the αCGRP polymerization.

Tracking the amyloidogenic core of IAPP amyloid fibrils: Insights from micro-Raman spectroscopy.

Human islet amyloid polypeptide (hIAPP) is the major protein component of extracellular amyloid deposits, located in the islets of Langerhans, a hallmark of type II diabetes. The underlying mechanisms of IAPP aggregation have not yet been clearly defined, although the highly amyloidogenic sequence of the protein has been extensively studied. Several segments have been highlighted as aggregation-prone regions (APRs), with much attention focused on the central 8-17 and 20-29 stretches. In this work, we employ micro-Raman spectroscopy to identify specific regions that are contributing to or are excluded from the amyloidogenic core of IAPP amyloid fibrils. Our results demonstrate that both the N-terminal region containing a conserved disulfide bond between Cys residues at positions 2 and 7, and the C-terminal region containing the only Tyr residue are excluded from the amyloid core. Finally, by performing detailed aggregation assays and molecular dynamics simulations on a number of IAPP variants, we demonstrate that point mutations within the central APRs contribute to the reduction of the overall amyloidogenic potential of the protein but do not completely abolish the formation of IAPP amyloid fibrils.

Unraveling the aggregation propensity of human insulin C-peptide.

Over the last 20 years, proinsulin C-peptide emerged as an important player in various biological events. Much time and effort has been spent in exploring all functional features of C-peptide and recording its implications in Diabetes mellitus. Only a few studies, though, have addressed C-peptide oligomerization and link this procedure with Diabetes. The aim of our work was to examine the aggregation propensity of C-peptide, utilizing Transmission Electron Microscopy, Congo Red staining, ATR-FTIR, and X-ray fiber diffraction at a 10 mg ml-1 concentration. Our experimental work clearly shows that C-peptide self-assembles into amyloid-like fibrils and therefore, the aggregation propensity of C-peptide is a characteristic novel feature that should be related to physiological and also pathological conditions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 108: 1-8, 2017.

Exploring Amyloidogenicity of Clusterin: A Structural and Bioinformatics Analysis.

Clusterin, a multitasking glycoprotein, is a protein highly conserved amongst mammals. In humans, Clusterin is mainly a secreted protein, described as an extracellular chaperone with the capability of interacting with a broad spectrum of molecules. In neurodegenerative diseases, such as Alzheimer's disease, it is an amyloid associated protein, co-localized with fibrillar deposits in amyloid plaques in systemic or localized amyloidoses. An 'aggregation-prone' segment (NFHAMFQ) was located within the Clusterin α-chain sequence using AMYLPRED, a consensus method for the prediction of amyloid propensity, developed in our lab. This peptide was synthesized and was found to self-assemble into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, Attenuated Total Reflectance Fourier-Transform Spectroscopy and Congo red staining studies reveal. All experimental results verify that this human Clusterin peptide-analogue, possesses high aggregation potency. Additional computational analysis highlighted novel and at the same time, unexplored features of human Clusterin.

Intrinsic aggregation propensity of the CsgB nucleator protein is crucial for curli fiber formation.

Several organisms exploit the extraordinary physical properties of amyloid fibrils forming natural protective amyloids, in an effort to support complex biological functions. Curli amyloid fibers are a major component of mature biofilms, which are produced by many Enterobacteriaceae species and are responsible, among other functions, for the initial adhesion of bacteria to surfaces or cells. The main axis of curli fibers is formed by a major structural subunit, known as CsgA. CsgA self-assembly is promoted by oligomeric nuclei formed by a minor curli subunit, known as the CsgB nucleator protein. Here, by implementing AMYLPRED2, a consensus prediction method for the identification of 'aggregation-prone' regions in protein sequences, developed in our laboratory, we have successfully identified potent amyloidogenic regions of the CsgB subunit. Peptide-analogues corresponding to the predicted 'aggregation-prone' segments of CsgB were chemically synthesized and studied, utilizing several biophysical techniques. Our experimental data indicate that these peptides self-assemble in solution, forming fibrils with characteristic amyloidogenic properties. Using comparative modeling techniques, we have developed three-dimensional models of both CsgA and CsgB subunits. Structural analysis revealed that the identified 'aggregation-prone' segments may promote gradual polymerization of CsgB. Briefly, our results indicate that the intrinsic self-aggregation propensity of the CsgB subunit, most probably has a pivotal role in initiating the formation of curli amyloid fibers by promoting the self-assembly process of the CsgB nucleator protein.

Identification of an amyloid fibril forming segment of human Pmel17 repeat domain (RPT domain).

Pmel17 is the major component of functional amyloid fibrils that have an important role during pigment deposition. Pmel17 polymerization is promoted within the mildly acidic conditions of melanosomes, organelles located in pigment-specific cells. A repeat domain (RPT domain) of Pmel17, rich in glutamic acid residues has been extensively associated with the formation of the fibrous matrix. Here, we examine the RPT domain of human Pmel17 in order to provide information on this mechanism. Specifically, we have identified an aggregation-prone peptide segment ((405) VSIVVLSGT(413) ), close to the C-terminal part of the RPT domain. Experimental results utilizing electron microscopy, X-ray fiber diffraction, Congo red staining and ATR FT-IR spectroscopy indicate that this peptide segment self-assembles forming fibrils with evident amyloidogenic properties. Conclusively, our results demonstrate that the (405) VSIVVLSGT(413) peptide segment possibly has an essential role in RPT domain fibrillogenesis.

A ß-solenoid model of the Pmel17 repeat domain: insights to the formation of functional amyloid fibrils.

Pmel17 is a multidomain protein involved in biosynthesis of melanin. This process is facilitated by the formation of Pmel17 amyloid fibrils that serve as a scaffold, important for pigment deposition in melanosomes. A specific luminal domain of human Pmel17, containing 10 tandem imperfect repeats, designated as repeat domain (RPT), forms amyloid fibrils in a pH-controlled mechanism in vitro and has been proposed to be essential for the formation of the fibrillar matrix. Currently, no three-dimensional structure has been resolved for the RPT domain of Pmel17. Here, we examine the structure of the RPT domain by performing sequence threading. The resulting model was subjected to energy minimization and validated through extensive molecular dynamics simulations. Structural analysis indicated that the RPT model exhibits several distinct properties of ß-solenoid structures, which have been proposed to be polymerizing components of amyloid fibrils. The derived model is stabilized by an extensive network of hydrogen bonds generated by stacking of highly conserved polar residues of the RPT domain. Furthermore, the key role of invariant glutamate residues is proposed, supporting a pH-dependent mechanism for RPT domain assembly. Conclusively, our work attempts to provide structural insights into the RPT domain structure and to elucidate its contribution to Pmel17 amyloid fibril formation.

Exploring the 'aggregation-prone' core of human Cystatin C: A structural study.

Amyloidogenic proteins like human Cystatin C (hCC) have been shown to form dimers and oligomers by exchange of subdomains of the monomeric proteins. Normally, the hCC monomer, a low molecular type 2 Cystatin, consists of 120 amino acid residues and functions as an inhibitor of cysteine proteases. The oligomerization of hCC is involved in the pathophysiology of a rare form of amyloidosis namely Icelandic hereditary cerebral amyloid angiopathy, in which an L68Q mutant is deposited as amyloid in brain arteries of young adults. In order to find the shortest stretch responsible to drive the fibril formation of hCC, we have previously demonstrated that the LQVVR peptide forms amyloid fibrils, in vitro (Tsiolaki et al., 2015). Predictions by AMYLPRED, an amyloidogenic determinant prediction algorithm developed in our lab, led us to synthesize and experimentally study two additional predicted peptides derived from hCC. Along with our previous findings, in this work, we reveal that these peptides self-assemble, in a similar way, into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies have shown. Further to our experimental results, all three peptides seem to have a fundamental contribution in forming the "aggregation-prone" core of human Cystatin C.