Experimental techniques for detecting and evaluating the amyloid fibrils.

Amyloid fibrils are insoluble proteins with intricate ß-sheet structures associated with various human diseases, including Parkinson's, Alzheimer's, and prion diseases. Proteins can form aggregates when their structure is misfolded, resulting in highly organized amyloid fibrils or amorphous aggregates. The formation of protein aggregates is a promising research field for mitigating diseases and the pharmaceutical and food industries. It is important to monitor and minimize the appearance of aggregates in these protein products. Several methods exist to assess protein aggregation, that includes from basic investigations to advanced biophysical techniques. Physicochemical parameters such as molecular weight, conformation, structure, and dimension are examined to study aggregation. There is an urgent need to develop methods for the detection of protein aggregation and amyloid fibril formation both in vitro and in vivo. This chapter focuses on a comprehensive discussion of the methods used to characterize and evaluate aggregates and amyloid fibrils.

The hidden world of protein aggregation.

Though the book's journey into The Hidden World of Protein Aggregation has come to an end, the search for knowledge, the development of healthier lives, and the discovery of nature's mysteries continue, promising new horizons and discoveries yet to be discovered. The intricacies of protein misfolding and aggregation remain a mystery in cellular biology, despite advances made in unraveling them. In this chapter, we will summarize the specific conclusions from the previous chapters and explore the persistent obstacles and unanswered questions that motivate scientists to pursue exploration of protein misfolding and aggregation.

Prediction of protein aggregation.

The scientific community is very interested in protein aggregation because of its involvement in several neurodegenerative diseases and its significance in industry. Remarkably, fibrillar aggregates are utilized naturally for constructing structural scaffolds or creating biological switches and may be intentionally designed to construct versatile nanomaterials. Consequently, there is a significant need to rationalize and predict protein aggregation. Researchers have developed various computational methodologies and algorithms to predict protein aggregation and understand its underlying mechanics. This chapter aims to summarize the significant advancements in computational methods, accessible resources, and prospective developments in the field of in silico research. We assess the existing computational tools for predicting protein aggregation propensities, detecting areas that are prone to sequential and structural aggregation, analyzing the effects of mutations on protein aggregation, or identifying prion-like domains.

Functional amyloids.

While amyloid has traditionally been viewed as a harmful formation, emerging evidence suggests that amyloids may also play a functional role in cell biology, contributing to normal physiological processes that have been conserved throughout evolution. Functional amyloids have been discovered in several creatures, spanning from bacteria to mammals. These amyloids serve a multitude of purposes, including but not limited to, forming biofilms, melanin synthesis, storage, information transfer, and memory. The functional role of amyloids has been consistently validated by the discovery of more functional amyloids, indicating a conceptual convergence. The biology of amyloids is well-represented by non-pathogenic amyloids, given the numerous ones already identified and the ongoing rate of new discoveries. In this chapter, functional amyloids in microorganisms, animals, and plants are described.

Therapeutic approaches in proteinopathies.

A family of maladies known as amyloid disorders, proteinopathy, or amyloidosis, are characterized by the accumulation of abnormal protein aggregates containing cross-ß-sheet amyloid fibrils in many organs and tissues. Often, proteins that have been improperly formed or folded make up these fibrils. Nowadays, most treatments for amyloid illness focus on managing symptoms rather than curing or preventing the underlying disease process. However, recent advances in our understanding of the biology of amyloid diseases have led to the development of innovative therapies that target the emergence and accumulation of amyloid fibrils. Examples of these treatments include the use of small compounds, monoclonal antibodies, gene therapy, and others. In the end, even if the majority of therapies for amyloid diseases are symptomatic, greater research into the biology behind these disorders is identifying new targets for potential therapy and paving the way for the development of more effective treatments in the future.

Protein aggregation: An overview.

In order for an ordered protein to perform its specific function, it must have a specific molecular structure. Information about this structure is encoded in the protein's amino acid sequence. The unique functional state is achieved as a result of a specific process, known as protein folding. However, as a result of partial or complete unfolding of the polypeptide chain, proteins may misfold and aggregate, leading to the formation of various aggregated structures, such as like amyloid aggregates with the cross-ß structure. A variety of cellular biological processes can be affected by protein aggregates that consume essential factors necessary for maintaining proteostasis, which leads to the proteostasis imbalance and further accumulation of protein aggregates, often resulting in age-related neurodegenerative disease progression and aging. However, in addition to their well-established pathological effects, amyloids also play various physiological roles, and many important biological processes involve such 'functional amyloids'. This chapter represents a brief overview of the protein aggregation phenomenon outlines a timeline provides of some key discoveries in this exciting field.

Pathways of amyloid fibril formation and protein aggregation.

The main cause of many neurodegenerative diseases and systemic amyloidoses is protein and peptide aggregation and the formation of amyloid fibrils. The study of aggregation mechanisms, the discovery and description of aggregate structures, and a comprehensive understanding of the molecular mechanisms of amyloid formation are of great importance for the diagnostic processes at the molecular level and for the development of therapeutic strategies to counter aggregation-associated disorders. Given that understanding protein misfolding phenomena is directly related to the protein folding process, we will briefly explain the protein folding mechanism and then discuss the important factors involved in protein aggregation. In the following, we review different mechanisms of amyloid formation and finally represent the current knowledge on how amyloid fibrils are formed based on kinetic and thermodynamic factors.

Biotechnological applications of amyloid fibrils.

Protein aggregates and amyloid fibrils have special qualities and are used in a variety of biotechnological applications. They are extensively employed in bioremediation, biomaterials, and biocatalysis. Because of their capacity to encapsulate and release pharmaceuticals and their sensitivity to certain molecules, respectively, they are also used in drug delivery and biosensor applications. They have also demonstrated potential in the domains of food and bioremediation. Additionally, amyloid peptides have drawn interest in biological applications, especially in the investigation of illnesses like Parkinson's and Alzheimer's. The unique characteristics of amyloid fibrils, namely their mechanical strength and ß-sheet structure, make them adaptable to a wide range of biotechnological uses. Even with their promise, one important factor to keep in mind before widely using modified amyloid materials is their potential toxicity. Thus, current research aims to overcome safety concerns while maximizing their potential.

Synthesis and evaluation of nanosystem containing chondroitinase ABCI based on hydroxyapatite.

The bacterial enzyme chondroitinase ABCI (chABCI), which has been isolated from Proteus Vulgaris, is crucial in the treatment of spinal cord injuries. However, due to its short lifespan, the maintenance and clinical application of this enzyme are very constrained. In this study, the immobilization of this enzyme on hydroxyapatite has been carried out and assessed with the aim of enhancing the characteristics and efficiency of chABCI. Hydroxyapatite particles (HAPs) are a potential candidate for drug-delivery carriers because of their excellent biocompatibility, shape controllability, and high adsorption. The use of the nanometer scale allows efficient access to the enzyme's substrate. It demonstrates important biological application capabilities in this way. Field emission gun-scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), infrared spectroscopy (FT-IR), in vitro release study, and cytotoxicity test were used to characterize the drug nanosystem's properties. According to the findings, electrostatic bindings was formed between charged groups of the enzyme and hydroxyapatite nanoparticles. The results also demonstrated that immobilized chABCI on hydroxyapatite has beneficial properties, such as more manageable drug release, minimal toxicity and side effects, and a high potential to enhance the efficacy of drug delivery and decrease the need for repeated injections.

Functional characterization of the dimeric form of PDGF-derived fusion peptide fabricated based on theoretical arguments.

A skin wound leads to the loss of skin integrity and the influx of pathogens into the tissue. Platelet-derived growth factors (PDGFs) are cytokines released from alpha granules during wound healing and interact with their cell surface receptors and activate signals involved in chemotaxis, growth, proliferation, and differentiation pathways. Due to the low stability of growth factors (GFs), a new peptide-derived PDGF-BB was designed, expressed in the Shuffle strain of E. coli, and purified by Ni-NTA agarose affinity column chromatography. The effect of fusion peptide was then evaluated on L929 fibroblast cells and animal models with skin lesions. In vitro, studies showed that the peptide led to an increase in the migration of fibroblast cells in the scratch assay. Its positive effect on wound healing was also observed in the skin-injured rats after 3, 7, and 12 days. A significant rise in neutrophils and granular tissue formation, re-epithelialization, angiogenesis, and collagen formation was exhibited on the third day of treatment when compared to the control group. The results showed that, despite reducing PDGF size, the fusion peptide was able to maintain at least some of the known functions attributed to full-length PDGF and showed positive results in wound healing.

Hippocampal tandem mass tag (TMT) proteomics analysis during kindling epileptogenesis in rat.

Epilepsy is a neurological disorder that remains difficult to treat due to the lack of a clear molecular mechanism and incomplete understanding of involved proteins. To identify potential therapeutic targets, it is important to gain insight into changes in protein expression patterns related to epileptogenesis. One promising approach is to analyze proteomic data, which can provide valuable information about these changes. In this study, to evaluate the changes in gene expression during epileptogenesis, LC-MC2 analysis was carried out on hippocampus during stages of electrical kindling in rat models. Subsequently, progressive changes in the expression of proteins were detected as a result of epileptogenesis development. In line with behavioral kindled seizure stages and according to the proteomics data, we described epileptogenesis phases by comparing Stage3 versus Control (S3/C0), Stage5 versus Stage3 (S5/S3), and Stage5 versus Control group (S5/C0). Gene ontology analysis on differentially expressed proteins (DEPs) showed significant changes of proteins involved in immune responses like Csf1R, Aif1 and Stat1 during S3/C0, regulation of synaptic plasticity like Bdnf, Rac1, CaMK, Cdc42 and P38 during S5/S3, and nervous system development throughout S5/C0 like Bdnd, Kcc2 and Slc1a3.There were also proteins like Cox2, which were altered commonly among all three phases. The pathway enrichment analysis of DEPs was also done to discover molecular connections between phases and we have found that the targets like Csf1R, Bdnf and Cox2 were analyzed throughout all three phases were highly involved in the PPI network analysis as hub nodes. Additionally, these same targets underwent changes which were confirmed through Western blotting. Our results have identified proteomic patterns that could shed light on the molecular mechanisms underlying epileptogenesis which may allow for novel targeted therapeutic strategies.

Rational design engineering of a more thermostable Sulfurihydrogenibium yellowstonense carbonic anhydrase for potential application in carbon dioxide capture technologies.

Implementing hyperthermostable carbonic anhydrases into CO2 capture and storage technologies in order to increase the rate of CO2 absorption from the industrial flue gases is of great importance from technical and economical points of view. The present study employed a combination of in silico tools to further improve thermostability of a known thermostable carbonic anhydrase from Sulfurihydrogenibium yellowstonense. Experimental results showed that our rationally engineered K100G mutant not only retained the overall structure and catalytic efficiency but also showed a 3 °C increase in the melting temperature and a two-fold improvement in the enzyme half-life at 85 °C. Based on the molecular dynamics simulation results, rearrangement of salt bridges and hydrogen interactions network causes a reduction in local flexibility of the K100G variant. In conclusion, our study demonstrated that thermostability can be improved through imposing local structural rigidity by engineering a single-point mutation on the surface of the enzyme.

Extracting Potential New Targets for Treatment of Adenoid Cystic Carcinoma using Bioinformatic Methods.

Background: Adenoid cystic carcinoma is a slow-growing malignancy that most often occurs in the salivary glands. Currently, no FDA-approved therapeutic target or diagnostic biomarker has been identified for this cancer. The aim of this study was to find new therapeutic and diagnostic targets using bioinformatics methods. Methods: We extracted the gene expression information from two GEO datasets (including GSE59701 and GSE88804). Different expression genes between adenoid cystic carcinoma (ACC) and normal samples were extracted using R software. The biochemical pathways involved in ACC were obtained by using the Enrichr database. PPI network was drawn by STRING, and important genes were extracted by Cytoscape. Real-time PCR and immunohistochemistry were used for biomarker verification. Results: After analyzing the PPI network, 20 hub genes were introduced to have potential as diagnostic and therapeutic targets. Among these genes, PLCG1 was presented as new biomarker in ACC. Furthermore, by studying the function of the hub genes in the enriched biochemical pathways, we found that insulin-like growth factor type 1 receptor and PPARG pathways most likely play a critical role in tumorigenesis and drug resistance in ACC and have a high potential for selection as therapeutic targets in future studies. Conclusion: In this study, we achieved the recognition of the pathways involving in ACC pathogenesis and also found potential targets for treatment and diagnosis of ACC. Further experimental studies are required to confirm the results of this study.

Improvement of thermal-stability of chondroitinase ABCI immobilized on graphene oxide for the repair of spinal cord injury.

Spinal cord injury healing has been shown to be aided by chondroitinase ABC I (cABCI) treatment. The transport of cABCI to target tissues is complicated by the enzyme's thermal instability; however, cABCI may be immobilized on nanosheets to boost stability and improve delivery efficiency. This investigation's goal was to assess the immobilization of cABC I on graphene oxide (GO). for this purpose, GO was produced from graphene using a modified version of Hummer's process. the immobilization of cABC I on GO was examined using SEM, XRD, and FTIR. The enzymatic activity of cABC I was evaluated in relation to substrate concentration. The enzyme was then surface-adsorption immobilized on GO, and its thermal stability was examined. As compared to the free enzyme, the results showed that the immobilized enzyme had a greater Km and a lower Vmax value. The stability of the enzyme was greatly improved by immobilization at 20, 4, 25, and 37 °C. For example, at 37 °C, the free enzyme retained 5% of its activity after 100 min, while the immobilized one retained 30% of its initial activity. The results showed, As a suitable surface for immobilizing cABC I, GO nano sheets boost the enzyme's stability, improving its capability to support axonal regeneration after CNC damage and guard against fast degradation.

The effect of Nrf2 deletion on the proteomic signature in a human colorectal cancer cell line.

BACKGROUND: Colorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated. METHODS: The influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2-, proteomic analysis, realtime PCR and western blotting were performed. RESULTS: In the absence of Nrf2 (Nrf2-), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2- cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2- cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production. CONCLUSIONS: Our results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.

SPR Analysis of SUMO-Murine Rap1-Interacting Factor 1 C-Terminal Domain Interaction with G4.

One of the advantages of surface plasmon resonance is its sensitivity and real-time analyses performed by this method. These characteristics allow us to further investigate the interactions of challenging proteins like Rap1-interacting factor 1 (Rif1). Rif1 is a crucial protein responsible for regulating different cellular processes including DNA replication, repair, and transcription. Mammalian Rif1 is yet to be fully characterized, partly because it is predicted to be intrinsically disordered for a large portion of its polypeptide. This protein has recently been the target of research as a potential biomarker in many cancers. Therefore, finding its most potent interacting partner is of utmost importance. Previous studies showed Rif1's affinity towards structured DNAs and amongst them, T6G24 was superior. Recent studies have shown mouse Rif1 (muRif1) C-terminal domain's (CTD) role in binding to G-quadruplexes (G4). There were many concerns in investigating the Rif1 and G4 interaction, which can be minimized using SPR. Therefore, for the first time, we have assessed its binding with G4 at nano-molar concentrations with SPR which seems to be crucial for its binding analyses. Our results indicate that muRif1-CTD has a high affinity for this G4 sequence as it shows a very low KD (6 ± 1 nM).

Characterization of Interaction of the MBP-Tagged MuRif1-C-Terminal Domain with G-Quadruplex DNA by SPR.

One of the main players in the cell-specific replication timing pattern is Rap1 interacting factor-1 (Rif1). Rif1 protein consists of N-terminal and C-terminal domains and an intrinsically disordered region in between. It has been suggested that both N- and C-termini of Rif1 are capable of binding to DNA with particularly high affinity to cruciform DNA structures. In the present study, we expressed, solubilized, and purified the maltose-binding protein-tagged murine Rif1 C-terminal domain (MBP-muRif1-CTD). Biological activity of the purified protein was assessed by the electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). Our results show that the MBP-muRif1-CTD binds G-quadruplex (G4) structure with high affinity (KD 19.0 ± 0.8 nM), as was previously suggested. This study is the first step in investigation of the interaction of MBP-Profinity eXact-muRif1-CTD and G4 by SPR.

CD38 and MGluR1 as possible signaling molecules involved in epileptogenesis: A potential role for NAD+ homeostasis.

In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD+) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD+ degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy: potential targets in calcium regulatory network.

Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca2+ homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.