Using silver nanoparticle-decorated whey protein isolate amyloid fibrils to modify the electrode surface used for electrochemical detection of para-nitrophenol.

Due to their organized structures, remarkable stiffness, and nice biocompatibility and biodegradability, amyloid fibrils serve as building blocks for versatile sustainable materials. Silver nanoparticles (AgNPs) are commonly used as the nano-catalysts for various electrochemical reactions. Given their large specific surface area and high surface energy, AgNPs exhibit high aggregation propensity, which hampers their electrocatalytic performance. Food protein wastes have been identified to be associated with climate change and environmental impacts, and a surplus of whey proteins in dairy industries causes high biological and chemical demands, and greenhouse gas emissions. This study is aimed at constructing sustainable electrode surface modifiers using AgNP-deposited whey protein amyloid fibrils (AgNP/WPI-AFs). AgNP/WPI-AFs were synthesized and characterized via spectroscopic techniques, electron microscopy, and X-ray diffraction. Next, the electrocatalytic performance of AgNP/WPI-AF modified electrode was assessed via para-nitrophenol (p-NP) reduction combined with various electrochemical analyses. Moreover, the reaction mechanism of p-NP electrocatalysis on the surface of AgNP/WPI-AF modified electrode was investigated. The detection range, limit of detection, sensitivity, and selectivity of the AgNP/WPI-AF modified electrode were evaluated accordingly. This work not only demonstrates an alternative for whey valorization but also highlights the feasibility of using amyloid-based hybrid materials as the electrode surface modifier for electrochemical sensing purposes.

Developing targeted drug delivery carriers for breast cancer using glutathione-sensitive doxorubicin-coupled glycated bovine serum albumin nanoparticles.

Incorporation of the nano-based carriers into drug delivery provides a promising alternative to overcome the limitations of the conventional chemotherapy. Doxorubicin (DOXO) is an effective chemotherapeutic drug widely used in chemotherapy for breast cancer treatment. A globular protein bovine serum albumin (BSA) holds great potential as carriers in pharmaceutical applications. This work is aimed at developing the DOXO-coupled glycated BSA nanoparticles via desolvation method for improving the capability of targeting the GLUT5 transporters over-expressed on breast cancer cells. Fructosamine assay and Fourier transform infrared spectroscopy were employed to determine the content of fructosamine structure and structural changes on the surfaces of nanoparticles, respectively. Additionally, the synthesized BSA nanoparticles were further characterized by electron microscopy and dynamic light scattering. Results revealed that the DOXO-coupled glycated BSA nanoparticles were spherically shaped with a hydrodynamic diameter of ~60.74 nm and a ζ-potential of ~ - 42.20 mV. Moreover, the DOXO release behavior of as-synthesized DOXO-coupled glycated BSA nanoparticles was examined under different conditions. Finally, the DOXO-coupled glycated BSA nanoparticles were found to exhibit cytotoxicity toward both MCF-7 and MDA-MB-231 cells. Our findings evidently suggested that the drug-coupled glycated BSA nanoparticles serve as the potential candidates for targeted drug delivery platform used in breast cancer therapy.

Application of Amyloid-Based Hybrid Membranes in Drug Delivery.

The properties of amyloid fibrils, e.g., unique structural characteristics and superior biocompatibility, make them a promising vehicle for drug delivery. Here, carboxymethyl cellulose (CMC) and whey protein isolate amyloid fibril (WPI-AF) were used to synthesize amyloid-based hybrid membranes as vehicles for the delivery of cationic and hydrophobic drugs (e.g., methylene blue (MB) and riboflavin (RF)). The CMC/WPI-AF membranes were synthesized via chemical crosslinking coupled with phase inversion. The zeta potential and scanning electron microscopy results revealed a negative charge and a pleated surface microstructure with a high content of WPI-AF. FTIR analysis showed that the CMC and WPI-AF were cross-linked via glutaraldehyde and the interacting forces between membrane and MB or RF was found to be electrostatic interaction and hydrogen bonding, respectively. Next, the in vitro drug release from membranes was monitored using UV-vis spectrophotometry. Additionally, two empirical models were used to analyze the drug release data and relevant rate constant and parameters were determined accordingly. Moreover, our results indicated that in vitro drug release rates depended on the drug-matrix interactions and transport mechanism, which could be controlled by altering the WPI-AF content in membrane. This research provides an excellent example of utilizing two-dimensional amyloid-based materials for drug delivery.

Silver nanoparticle-deposited whey protein isolate amyloid fibrils as catalysts for the reduction of methylene blue.

The unique structural characteristics and superior biocompatibility make the protein nanofibers promising immobilization platforms/substrates for catalysts/enzymes. Metal nanoparticles have been employed as the catalysts in industries due to their excellent catalytic activity and stability, whereas their high surface energy leads to nanoparticle aggregation, thereby hampering their catalytic performance. Here, amyloid fibril (AF) derived from whey protein isolate (WPI) was chosen as the support of silver nanoparticles (AgNP) and utilized for the catalytic reduction of methylene blue (MB). The one-dimensional amyloid-based hybrid materials (AgNP/WPI-AF) were first synthesized via chemical or photochemical route. The characterization of AgNP/WPI-AF by UV-vis spectrophotometry and electron microscopy revealed that the sizes of AgNP on WPI-AF's surface ranged from 2 to 30 nm. Next, the catalytic performances of AgNP/WPI-AF prepared by various routes for MB degradation were investigated. Additionally, the kinetic data were analyzed using two different models and the apparent rate constants and thermodynamic parameters were further determined accordingly. Moreover, the reusability of AgNP/WPI-AF was assessed by monitoring the percentage removal of MB over consecutive filtering cycles. Our results indicated that Langmuir-Hinshelwood-type mechanism better described the catalytic MB reduction using AgNP/WPI-AF. This work provides a nice example of application of nanoparticle-amyloid fibril composite materials for catalysis.

Insights to Human γD-Crystallin Unfolding by NMR Spectroscopy and Molecular Dynamics Simulations.

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis.

Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Characterization of Androgen Receptor Complex Associated Protein (ARCAP) in hepatocellular carcinoma and liver.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks many tasks in clinical oncology due to possibly developing a general tumor in men and, usually lead to malignant to death within years. Researches had reported about major factors for being HCC was male sex and HCC associated with cirrhosis in childhood was found more common in males than females. In certain mouse strains as studied, breeding with testosterone significantly increases the development of HCC. Furthermore, castration of male mice diminished the frequency of the development of liver tumors. Meanwhile male hepatitis B virus transgenic mice have a greater occurrence of HCC than females. METHODS: We apply degenerate priming PCR to observe the expression of various steroid receptors in livers. Yeast-two hybrid screening to search a novel RNA fragment helps to find a new full-length gene by RACE experiment. RT-PCR is applied to detect various expressions in tissues and cell lines. In situ hybridization detects DNA in Chromosome mapping. GFP-constructs transfection proves the gene localization in cells. Immunoprecipitation pulldown assay verifies protein interaction. Gene transfection followed with luciferase assay demonstrates the interaction of genes within cellular signaling. Genomic alignment analysis for observing sequences data perform from NCBI database website (http://www.ncbi.nim.nih.gov/genebank/). RESULTS: The androgen receptor (AR) expression level is found at the highest level among the steroid receptors families detected in liver tumors. By yeast-two hybrid screening, we cloned an Androgen Receptor Complex Associated Protein (ARCAP), of 95 Kd in molecular weight and its cDNA. ARCAP locates at Chromosome 1. Our findings indicate ARCAP is highly expressed in hepatoma cell lines and liver tumors and their adjacent tumors as observed. Yeast two-hybrid assay and in vitro immunoprecipitation assays demonstrated an interaction between AR and ARCAP. CONCLUSION: We aim to search for different types and levels of steroid receptors expressed within human HCCs and in the adjacent liver tissues. To verify possible molecular mechanisms by which AR might affect hepatoma cells, we had characterized a novel protein ARCAP which functions as a coregulator to interact with AR within liver. The ligand-dependent AR with its cofactor, ARCAP, can induce a signal cascade by transactivation.

USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer.

USP7, one of the most abundant ubiquitin-specific proteases (USP), plays multifaceted roles in many cellular events, including oncogenic pathways. Accumulated studies have suggested that USP7, through modulating the MDM2/MDMX-p53 pathway, is a promising target for cancer treatment; however, little is known about the function of USP7 in p53-deficient tumors. Here we report that USP7 regulates the autoregulation of SMAD3, a key regulator of transforming growth factor ß (TGFß) signaling, that represses the cell progression of p53-deficient lung cancer. CRISPR/Cas9-mediated inactivation of USP7 in p53-deficient lung cancer H1299 line resulted in advanced cell proliferation in vitro and in xenograft tumor in vivo. Genome-wide analyses (ChIP-seq and RNA-seq) of USP7 KO H1299 cells reveal a dramatic reduction of SMAD3 autoregulation, including decreased gene expression and blunted function of associated super-enhancer (SE). Furthermore, biochemical assays show that SMAD3 is conjugated by mono-ubiquitin, which negatively regulates the DNA-binding function of SMAD3, in USP7 KO cells. In addition, cell-free and cell-based analyses further demonstrate that the deubiquitinase activity of USP7 mediates the removal of mono-ubiquitin from SMAD3 and facilitates the DNA-binding of SMAD3-SMAD4 dimer at SMAD3 locus, and thus enhance the autoregulation of SMAD3. Collectively, our study identified a novel mechanism by which USP7, through catalyzing the SMAD3 de-monoubiquitination, facilitates the positive autoregulation of SMAD3, and represses the cancer progression of p53-deficient lung cancer.

Examining the effect of bovine serum albumin on the properties and drug release behavior of ß-lactoglobulin-derived amyloid fibril-based hydrogels.

Herein, we report the use of ß-lactoglobulin (ß-LG) combined with bovine serum albumin (BSA) for the preparation of amyloid-based hydrogels with aim of delivering riboflavin. The incorporation of BSA enhanced ß-LG fibrillogenesis and protected ß-LG fibrils from losing fibrillar structure due to the pH shift. The mechanical properties of hydrogels were observed to be positively correlated with the number of amyloid fibrils. While the addition of BSA induced amyloid fibril formation, its presence between the fibril chains interfered with the entanglement of fibril chains, thus adversely affecting the hydrogels' mechanical properties. Hydrogels' surface microstructure became more compact as the number of amyloid fibrils rose and the presence of BSA could improve hydrogels' surface homogeneity. In vitro riboflavin (RF) release rate was found to be correlated with the number of fibrils and BSA-RF binding affinity. However, when the digestive enzymes were present, the influence of BSA-RF affinity was alleviated due to enzymes' destructive and/or degradative effects on BSA and/or hydrogels, thus the release rate relied on the number of fibrils, which could be adjusted by the amount of BSA. Results indicate that the additional component, BSA, plays an important role in modulating the properties and functions of ß-LG fibril-based hydrogels.

Protection of human γD-crystallin protein from ultraviolet C-induced aggregation by ortho-vanillin.

Cataract is known as one of the leading causes of vision impairment worldwide. While the detailed mechanism of cataratogenesis remains unclear, cataract is believed to be correlated with the aggregation and/or misfolding of human ocular lens proteins called crystallins. A 173-residue structural protein human γD-crystallin is a major γ-crystallin protein in the human eye lens and associated with the development of juvenile and mature-onset cataracts. This work is aimed at investigating the effect of a small molecule, e.g., ortho-vanillin, on human γD-crystallin aggregation upon exposure to ultraviolet-C irradiation. According to the findings of right-angle light scattering, transmission electron microscopy, and gel electrophoresis, ortho-vanillin was demonstrated to dose-dependently suppress ultraviolet-C-triggered aggregation of human γD-crystallin. Results from the synchronous fluorescence spectroscopy, tryptophan fluorescence quenching, and molecular docking studies revealed the structural change of γD-crystallin induced by the interaction/binding between ortho-vanillin and protein. We believe the outcome from this work may contribute to the development of potential therapeutics for cataract.

Disulfiram and 6-Thioguanine synergistically inhibit the enzymatic activities of USP2 and USP21.

Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that disulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggesting an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like proteases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6-Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhibited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clinical applications.

Genomic variance of Open Reading Frames (ORFs) and Spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

BACKGROUND: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe pneumonia at December 2019. Since then, it has been wildly spread from Wuhan, China, to Asia, European, and United States to become the pandemic worldwide. Now coronavirus disease 2019 were globally diagnosed over 3 084 740 cases with mortality of 212 561 toll. Current reports variants are found in SARS-CoV-2, majoring in functional ribonucleic acid (RNA) to transcribe into structural proteins as transmembrane spike (S) glycoprotein and the nucleocapsid (N) protein holds the virus RNA genome; the envelope (E) and membrane (M) alone with spike protein form viral envelope. The nonstructural RNA genome includes ORF1ab, ORF3, ORF6, 7a, 8, and ORF10 with highly conserved information for genome synthesis and replication in ORF1ab. METHODS: We apply genomic alignment analysis to observe SARS-CoV-2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1); MN985325 (United States: WA, UW); MN996527 (China, C2); MT007544 (Australia: Victoria, A1); MT027064 (United States: CA, UC); MT039890 (South Korea, K1); MT066175 (Taiwan, T1); MT066176 (Taiwan, T2); LC528232 (Japan, J1); and LC528233 (Japan, J2) and Global Initiative on Sharing All Influenza Data database (https://www.gisaid.org). We adopt Multiple Sequence Alignments web from Clustalw (https://www.genome.jp/tools-bin/clustalw) and Geneious web (https://www.geneious.com. RESULTS: We analyze database by genome alignment search for nonstructural ORFs and structural E, M, N, and S proteins. Mutations in ORF1ab, ORF3, and ORF6 are observed; specific variants in spike region are detected. CONCLUSION: We perform genomic analysis and comparative multiple sequence of SARS-CoV-2. Large scaling sequence alignments trace to localize and catch different mutant strains in United possibly to transmit severe deadly threat to humans. Studies about the biological symptom of SARS-CoV-2 in clinic animal and humans will be applied and manipulated to find mechanisms and shield the light for understanding the origin of pandemic crisis.

Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4.

Phostensin (PTS) encoded by KIAA1949 is a protein phosphatase 1 (PP1)-binding protein. In order to explore the cellular functions of PTS, we have searched PTS-binding proteins by using co-immunoprecipitation in combination with shotgun proteomics. Here, we report two novel PTS-binding proteins, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS associated with EHD proteins was also observed in GST pull-down assays. Immunofluorescence microscopy demonstrated that the complex was co-localized at the endocytic vesicles. EHD proteins have been known to play a critical role in regulation of endocytic transport. Overexpression of PTS-ß can attenuate the endocytic trafficking of transferrin.

Hunting severe acute respiratory syndrome coronavirus 2 (2019 novel coronavirus): From laboratory testing back to basic research.

The rapid spread of coronavirus disease 2019 (COVID-19) in many countries causes citizens of daily inconvenience and even life-threat for elderly population. The invasion of the main pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; 2019 novel coronavirus [2019-nCoV]), into human body causes different levels of impact to various patients. One of the most important issues for COVID-19 is how to defend this virus with the ability to foresee the infected targets. Thus, we maintain the quarantined essentially as for as others saved from COVID-19. So far, the routine laboratory test to confirm whether infected by SARS-CoV-2/2019-nCoV or not is through real-time reverse transcription polymerase chain reaction (rRT-PCR; quantitative polymerase chain reaction [qPCR]) with certain sequence regions that recognize SARS-CoV-2/2019-nCoV RNA genome. The heavy loading of rRT-PCR (qPCR) machine and handling labor have tight-packed the instruments as well as the manpower almost in every country. Therefore, the alternative approaches are eagerly waiting to be developed. In this review article, we sort out some state-of-the-art novel approaches that might be applied for a fast, sensitive, and precise detection of SARS-CoV-2/2019-nCoV not only to help the routine laboratory testing but also to improve effective quarantine.

Novel biosensor platforms for the detection of coronavirus infection and severe acute respiratory syndrome coronavirus 2.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been causing respiratory diseases globally, damaging wide ranges of social-economic activities. This virus is transmitted through personal contact and possibly also through ambient air. Effective biosensor platforms for the detection of this virus and the related host response are in urgent demand. These platforms can facilitate routine diagnostic assays in certified clinical laboratories. They can also be integrated into point-of-care products. Furthermore, environmental biosensors can be designed to detect SARS-CoV-2 in the ambient air or in the intensive care ventilators. Here, we evaluate technical components of biosensors, including the biological targets of recognition, the recognition methods, and the signal amplification and transduction systems. Effective SARS-CoV-2 detectors can be designed by an adequate combination of these technologies.

Genomic analysis and comparative multiple sequences of SARS-CoV2.

BACKGROUND: China announced an outbreak of new coronavirus in the city of Wuhan on December 31, 2019; lash to now, the virus transmission has become pandemic worldwide. Severe cases from the Huanan Seafood Wholesale market in Wuhan were confirmed pneumonia with a novel coronavirus (2019-nCoV). Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies. Thus, we defined the correlation in 10 severe acute respiratory syndrome coronavirus (SARS-CoV2) sequences from different countries to analyze the genomic patterns of disease origin and evolution aiming for developing new control pandemic processes. METHODS: We apply genomic analysis to observe SARS-CoV2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1), MN985325 (USA: WA, UW), MN996527 (China, C2), MT007544 (Australia: Victoria, A1), MT027064 (USA: CA, UC), MT039890 (South Korea, K1), MT066175 (Taiwan, T1), MT066176 (Taiwan, T2), LC528232 (Japan, J1), and LC528233 (Japan, J2) for genomic sequence alignment analysis. Multiple Sequence Alignment by Clustalw (https://www.genome.jp/tools-bin/clustalw) web service is applied as our alignment tool. RESULTS: We analyzed 10 sequences from the National Center for Biotechnology Information (NCBI) database by genome alignment and found no difference in amino acid sequences within M and N proteins. There are two amino acid variances in the spike (S) protein region. One mutation found from the South Korea sequence is verified. Two possible "L" and "S" SNPs found in ORF1ab and ORF8 regions are detected. CONCLUSION: We performed genomic analysis and comparative multiple sequences of SARS-CoV2. Studies about the biological symptoms of SARS-CoV2 in clinic animals and humans will manipulate an understanding on the origin of pandemic crisis.

Conformational Characterization of Native and L17A/F19A-Substituted Dutch-Type ß-Amyloid Peptides.

Some mutations which occur in the α/ß-discordant region (resides 15 to 23) of ß-amyloid peptide (Aß) lead to familial Alzheimer's disease (FAD). In vitro studies have shown that these genetic mutations could accelerate Aß aggregation. We recently showed that mutations in this region could alter the structural propensity, resulting in a different aggregative propensity of Aß. Whether these genetic mutations display similar effects remains largely unknown. Here, we characterized the structural propensity and aggregation kinetics of Dutch-type Aß40 (Aß40(E22Q)) and its L17A/F19A-substituted mutant (Aß40(L17A/F19A/E22Q)) using circular dichroism spectroscopy, nuclear magnetic spectroscopy, and thioflavin T fluorescence assay. In comparison with wild-type Aß40, we found that Dutch-type mutation, unlike Artic-type mutation (E22G), does not reduce the α-helical propensity of the α/ß-discordant region in sodium dodecyl sulfate micellar solution. Moreover, we found that Aß40(L17A/F19A/E22Q) displays a higher α-helical propensity of the α/ß-discordant region and a slower aggregation rate than Aß40(E22Q), suggesting that the inhibition of aggregation might be via increasing the α-helical propensity of the α/ß-discordant region, similar to that observed in wild-type and Artic-type Aß40. Taken together, Dutch-type and Artic-type mutations adopt different mechanisms to promote Aß aggregation, however, the L17A/F19A mutation could increase the α-helical propensities of both Dutch-type and Artic-type Aß40 and inhibit their aggregation.

Exploring the effects of methylene blue on amyloid fibrillogenesis of lysozyme.

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Characterization of the interactions between inhibitor-1 and recombinant PP1 by NMR spectroscopy.

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.