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1.
J Chem Phys ; 157(5): 055101, 2022 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-35933209

RESUMO

The formation of the fibrillar structure of amyloid proteins/peptides is believed to be associated with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Since the rate of aggregation can influence neurotoxicity, finding the key factors that control this rate is of paramount importance. It was recently found that the rate of protein aggregation is related to the mechanical stability of the fibrillar structure such that the higher the mechanical stability, the faster the fibril is formed. However, this conclusion was supported by a limited dataset. In this work, we expand the previous study to a larger dataset, including the wild type of Aß42 peptide and its 20 mutants, the aggregation rate of which was measured experimentally. By using all-atom steered molecular dynamics (SMD) simulations, we can assess the mechanical stability of the fibril structure, which is characterized by the rupture force, pulling work, and unbinding free energy barrier. Our result confirms that mechanical stability is indeed related to the aggregation rate. Since the estimation of the aggregation rate using all-atom simulations is almost forbidden by the current computational capabilities, our result is useful for predicting it based on information obtained from fast SMD simulations for fibrils.


Assuntos
Doença de Alzheimer , Agregados Proteicos , Doença de Alzheimer/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Estabilidade Proteica
2.
Methods Mol Biol ; 2538: 35-54, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35951292

RESUMO

Bacterial amyloids decorate the cell surface of many bacteria by forming functional amyloid fibers. These amyloids have structural and biochemical similarities with many disease-related amyloids in eukaryotes. Amyloid aggregation starts at the individual monomer level, and the end product is the amyloid fibril. The process of amyloid aggregation involves a continuous increase of the aggregate size, and therefore size is a critical parameter to measure in aggregation experiments. Also, our understanding of the aggregation process, and our ability to design interventions, can benefit from a measurement of the conformational dynamics of proteins undergoing aggregation. Fluorescence correlation spectroscopy (FCS) is perhaps the most sensitive and rapid technique available currently for this purpose. It can measure the average size and the size distribution of molecules and aggregates down to sub-nm length scales and can also measure fast nanosecond time-scale conformational dynamics, all in an equilibrium solution. FCS achieves this by measuring the fluorescence intensity fluctuations of freely diffusing protein molecules in an optically defined microscopic probe volume in a solution. Here, we present a set of instructions for effectively measuring the size and dynamics of amyloid aggregates with FCS.


Assuntos
Amiloide , Proteínas Amiloidogênicas , Amiloide/química , Peptídeos beta-Amiloides/química , Difusão , Agregados Proteicos , Espectrometria de Fluorescência/métodos
3.
Cell Death Dis ; 13(8): 688, 2022 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-35933423

RESUMO

cAMP Responsible Element Binding Protein (CREB) is an evolutionarily conserved transcriptional factor that regulates cell growth, synaptic plasticity and so on. In this study, we unexpectedly found proteasome inhibitors, such as MLN2238, robustly increase CREB activity in adult flies through a large-scale compound screening. Mechanistically, reactive oxidative species (ROS) generated by proteasome inhibition are required and sufficient to promote CREB activity through c-Jun N-terminal kinase (JNK). In 293 T cells, JNK activation by MLN2238 is also required for increase of CREB phosphorylation at Ser133. Meanwhile, transcriptome analysis in fly intestine identified a group of genes involved in redox and proteostatic regulation are augmented by overexpressing CRTC (CREB-regulated transcriptional coactivator). Intriguingly, CRTC overexpression in muscles robustly restores protein folding and proteasomal activity in a fly Huntington's disease (HD) model, and ameliorates HD related pathogenesis, such as protein aggregates, motility, and lifespan. Moreover, CREB activity increases during aging, and further enhances its activity can suppress protein aggregates in aged muscles. Together, our results identified CRTC/CREB downstream ROS/JNK signaling as a conserved sensor to tackle oxidative and proteotoxic stresses. Boosting CRTC/CREB activity is a potential therapeutic strategy to treat aging related protein aggregation diseases.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Drosophila , Animais , Proteínas de Transporte/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Drosophila/genética , Drosophila/metabolismo , Fosforilação , Agregados Proteicos , Espécies Reativas de Oxigênio/metabolismo
4.
Elife ; 112022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35942699

RESUMO

Dystonin (DST), which encodes cytoskeletal linker proteins, expresses three tissue-selective isoforms: neural DST-a, muscular DST-b, and epithelial DST-e. DST mutations cause different disorders, including hereditary sensory and autonomic neuropathy 6 (HSAN-VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-related diseases has been unclear. Because DST-b contains all of the DST-a-encoding exons, known HSAN-VI mutations could affect both DST-a and DST-b isoforms. To investigate the specific function of DST-b in striated muscles, we generated a Dst-b-specific mutant mouse model harboring a nonsense mutation. Dst-b mutant mice exhibited late-onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also found nuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations as a unique pathological hallmark in Dst-b mutation-induced cardiomyopathy. RNA-sequencing analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico analysis identified DST-b alleles with nonsense mutations in populations worldwide, suggesting that some unidentified hereditary myopathy and cardiomyopathy are caused by DST-b mutations. Here, we demonstrate that the Dst-b isoform is essential for long-term maintenance of striated muscles.


Assuntos
Cardiomiopatias , Distonina/genética , Neuropatias Hereditárias Sensoriais e Autônomas , Doenças Musculares , Animais , Cardiomiopatias/genética , Distonina/metabolismo , Camundongos , Mutação , Agregados Proteicos , Isoformas de Proteínas/genética
5.
Nat Commun ; 13(1): 4692, 2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948542

RESUMO

Huntington's disease is a neurodegenerative disease caused by an expanded polyQ stretch within Huntingtin (HTT) that renders the protein aggregation-prone, ultimately resulting in the formation of amyloid fibrils. A trimeric chaperone complex composed of Hsc70, DNAJB1 and Apg2 can suppress and reverse the aggregation of HTTExon1Q48. DNAJB1 is the rate-limiting chaperone and we have here identified and characterized the binding interface between DNAJB1 and HTTExon1Q48. DNAJB1 exhibits a HTT binding motif (HBM) in the hinge region between C-terminal domains (CTD) I and II and binds to the polyQ-adjacent proline rich domain (PRD) of soluble as well as aggregated HTT. The PRD of HTT represents an additional binding site for chaperones. Mutation of the highly conserved H244 of the HBM of DNAJB1 completely abrogates the suppression and disaggregation of HTT fibrils by the trimeric chaperone complex. Notably, this mutation does not affect the binding and remodeling of any other protein substrate, suggesting that the HBM of DNAJB1 is a specific interaction site for HTT. Overexpression of wt DNAJB1, but not of DNAJB1H244A can prevent the accumulation of HTTExon1Q97 aggregates in HEK293 cells, thus validating the biological significance of the HBM within DNAJB1.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Amiloide/química , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Agregados Proteicos
6.
Acta Myol ; 41(2): 59-75, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35832504

RESUMO

Bcl2-associated athanogene 3 (BAG3) is a multifunctional cochaperone responsible for protein quality control within cells. BAG3 interacts with chaperones HSPB8 and Hsp70 to transport misfolded proteins to the Microtubule Organizing Center (MTOC) and degrade them in autophagosomes in a process known as Chaperone Assisted Selective Autophagy (CASA). Mutations in the second conserved IPV motif of BAG3 are known to cause Dilated Cardiomyopathy (DCM) by inhibiting adequate removal of non-native proteins. The proline 209 to leucine (P209L) BAG3 mutant in particular causes the aggregation of BAG3 and misfolded proteins as well as the sequestration of essential chaperones. The exact mechanisms of protein aggregation in DCM are unknown. However, the similar presence of insoluble protein aggregates in Charcot-Marie-Tooth disease type 2 (CMT2) induced by the proline 182 to leucine (P182L) HSPB1 mutant points to a possible avenue for future research: IPV motif. In this review, we summarize the molecular mechanisms of CASA and the currently known pathological effects of mutated BAG3 in DCM. Additionally, we will provide insight on the importance of the IPV motif in protein aggregation by analyzing a potential association between DCM and CMT2.


Assuntos
Cardiomiopatia Dilatada , Doença de Charcot-Marie-Tooth , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Dilatada/genética , Doença de Charcot-Marie-Tooth/genética , Humanos , Leucina , Prolina , Agregados Proteicos
7.
ACS Chem Neurosci ; 13(15): 2281-2287, 2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35856925

RESUMO

A strong association between protein aggregation and human diseases (such as Alzheimer's, Parkinson's, and Huntington's disease) is well demonstrated. Misfolding and aggregation of p53, a central transcriptional mediator, has been revealed by various experimental evidence in different types of cancers. Aggregation studies focusing on different p53 domains, mostly, the central core domain and its mutants under the influence of various environmental conditions, and the p53 transactivation domain (TAD) (1-63) have been reported. However, the specific subdomains responsible for p53 aggregation are not known. p53 TADs interact with diverse cellular factors to modulate the function of p53 and elicit appropriate cellular responses under different stress conditions. In this study, the aggregation of the p53 TAD2 domain (38-61) has been studied in isolation. The aggregates were generated in vitro under acidic pH conditions after in silico scoring for amyloidogenic tendency and characterized using dye-based assays (ThT and bis-ANS fluorescence), CD spectroscopy, and microscopy (scanning electron microscoy, transmission electron microscopy, and atomic force microscopy). It was observed that p53 TAD2 forms characteristic ß-sheet-rich amyloid-like fibrils. Via a reductionist approach, this study highlights the nature of p53 TAD2 domain (38-61) aggregation.


Assuntos
Amiloidose , Proteína Supressora de Tumor p53 , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Humanos , Agregados Proteicos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Molecules ; 27(13)2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35807552

RESUMO

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and ß-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas de Ligação a DNA , Degeneração Lobar Frontotemporal , Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Agregados Proteicos , Conformação Proteica em Folha beta , Domínios Proteicos , Dobramento de Proteína
9.
STAR Protoc ; 3(3): 101571, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35880124

RESUMO

The loss of protein homeostasis results in cytotoxic protein aggregates, a common hallmark of aging and neurological diseases. Here, we present an adjusted filter-trapping assay protocol to detect global aggregated proteins in human cell lines, via a high-sensitive protein staining method. This protocol also details an alternative approach to monitor specific protein aggregates trapped in the filter membrane, by subsequent immunoblotting of ectopically expressed and endogenous proteins. For complete details on the use and execution of this protocol, please refer to Chhipi-Shrestha et al. (2022).


Assuntos
Agregados Proteicos , Proteínas , Bioensaio , Linhagem Celular , Humanos , Coloração e Rotulagem
11.
Food Funct ; 13(15): 8158-8167, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35820177

RESUMO

Introducing membrane filtration steps into infant milk formula (IMF) manufacture can partly preserve native whey proteins in the final products. In this study, the IMF produced by membrane filtration (MEM-IMF) and conventional heat treatment (HT-IMF) were compared by using a novel semi-dynamic infant in vitro digestion method. MEM-IMF exhibited a fragmented curd during gastric digestion, and confocal laser light microscopy showed that protein aggregates had disassociated from the fat droplets within 93 min in the MEM-IMF digesta. In contrast, the digesta of HT-IMF showed a more extensive curd formation and denser protein aggregates, which remained intact until the end of gastric digestion. Molecular weight profiles and the primary amine assay suggested that protein degradation and peptide release were faster in the MEM-IMF. In conclusion, the presence of native whey protein in the IMF altered the gastric digestion kinetics by changing coagulation and formation of aggregates, potentially accelerating the rate of gastric emptying in vivo.


Assuntos
Temperatura Alta , Fórmulas Infantis , Animais , Digestão , Humanos , Lactente , Fórmulas Infantis/química , Leite , Agregados Proteicos , Proteínas do Soro do Leite
12.
Anal Chem ; 94(29): 10320-10328, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35848328

RESUMO

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism─with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers─all confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications─all while retaining the speed and compatibility of SP3.


Assuntos
Proteoma , Proteômica , Fenômenos Magnéticos , Agregados Proteicos , Proteoma/análise , Reprodutibilidade dos Testes , Solventes
13.
Int J Biochem Cell Biol ; 149: 106258, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35777599

RESUMO

Limited treatment options and research in understanding the pathomechanisms of rare diseases has raised concerns about their therapeutic development. One such poorly understood ultra-rare neuromuscular disorder is GNE Myopathy (GNEM) which is caused due to mutation in key sialic acid biosynthetic enzyme, GNE. Treatment with sialic acid or its derivatives/precursors slows the disease progression, but curative strategies need to be explored further. Pathologically, muscle biopsy samples of GNEM patients reveal rimmed vacuole formation due to aggregation of ß-amyloid, Tau, presenilin proteins with unknown mechanism. The present study aims to understand the mechanism of protein aggregate formation in GNE mutant cells to decipher role of chaperones in disease phenotype. The pathologically relevant GNE mutations expressed as recombinant proteins in HEK cells was used as a model system for GNEM to estimate extent of protein aggregation. We identified HSP70, a chaperone, as binding partner of GNE. Downregulation of HSP70 with altered BAG3, JNK, BAX expression levels was observed in GNE mutant cells. The cell apoptosis was observed in GNE mutation specific manner. An activator of HSP70 chaperone, BGP-15, rescued the phenotypic defects due to GNE mutation, thereby, reducing protein aggregation significantly. The results were further validated in rat skeletal muscle cell lines carrying single Gne allele. Our study suggests that HSP70 activators can be a promising therapeutic target in the treatment of ultra-rare GNE Myopathy disease.


Assuntos
Miopatias Distais , Agregados Proteicos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Miopatias Distais/genética , Miopatias Distais/metabolismo , Miopatias Distais/patologia , Humanos , Chaperonas Moleculares/metabolismo , Músculo Esquelético/metabolismo , Mutação , Ácido N-Acetilneuramínico/metabolismo , Fenótipo
14.
Anal Chem ; 94(31): 10949-10958, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35877130

RESUMO

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.


Assuntos
COVID-19 , Agregados Proteicos , Humanos , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , SARS-CoV-2
15.
Int J Biol Macromol ; 216: 42-51, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35779650

RESUMO

Previously, N-acetyl-l-arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (Tm) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the Tm by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (Tagg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG.


Assuntos
Agregados Proteicos , Óleos de Silicone , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Etanercepte/química , Feminino , Imunidade Inata , Imunoglobulinas Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Óleos de Silicone/química
16.
EMBO J ; 41(16): e111700, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35791595

RESUMO

A hallmark of age-related neurodegenerative diseases is the presence of highly stable protein aggregates, also known as amyloid fibres. As these fibres are strongly associated with disease, it is thought that clearance of these fibres could delay or prevent disease progression. In this issue of The EMBO Journal, Beton et al unravel how the Hsc70/DNAJB1/Apg2 disaggregase machinery disassembles amyloid fibres, using α-synuclein fibrils implicated in Parkinson's Disease as a model substrate.


Assuntos
Amiloide , Doença de Parkinson , Amiloide/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Cinética , Doença de Parkinson/metabolismo , Agregados Proteicos , alfa-Sinucleína/metabolismo
17.
Molecules ; 27(15)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35897966

RESUMO

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-ß (Aß) characterized from experimental and theoretical approaches. Aß exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the ß-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aß amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures.


Assuntos
Amiloide , Agregados Proteicos , Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Conformação Proteica , Dobramento de Proteína
18.
Microbiologyopen ; 11(3): e1284, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35765185

RESUMO

Arsenic is a toxic metalloid that affects human health by causing numerous diseases and by being used in the treatment of acute promyelocytic leukemia. Saccharomyces cerevisiae (budding yeast) has been extensively utilized to elucidate the molecular mechanisms underlying arsenic toxicity and resistance in eukaryotes. In this study, we applied a genomic DNA overexpression strategy to identify yeast genes that provide arsenic resistance in wild-type and arsenic-sensitive S. cerevisiae cells. In addition to known arsenic-related genes, our genetic screen revealed novel genes, including PHO86, VBA3, UGP1, and TUL1, whose overexpression conferred resistance. To gain insights into possible resistance mechanisms, we addressed the contribution of these genes to cell growth, intracellular arsenic, and protein aggregation during arsenate exposure. Overexpression of PHO86 resulted in higher cellular arsenic levels but no additional effect on protein aggregation, indicating that these cells efficiently protect their intracellular environment. VBA3 overexpression caused resistance despite higher intracellular arsenic and protein aggregation levels. Overexpression of UGP1 led to lower intracellular arsenic and protein aggregation levels while TUL1 overexpression had no impact on intracellular arsenic or protein aggregation levels. Thus, the identified genes appear to confer arsenic resistance through distinct mechanisms but the molecular details remain to be elucidated.


Assuntos
Arsênio , Proteínas de Saccharomyces cerevisiae , Arsênio/metabolismo , Arsênio/toxicidade , Humanos , Agregados Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Anal Methods ; 14(24): 2350-2356, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35647934

RESUMO

In this paper, we present a robust and versatile method for developing non-label aptasensors with high sensitivity. Amyloid-like protein aggregates were facilely synthesized with the commonly used passivating agent bovine serum albumin (BSA) in developing biosensors, and the produced amyloid-like phase-transited BSA (PTB) exhibited excellent antifouling performances and robust interfacial adhesion with the electrode surface. In order to improve the detection sensitivity of electrochemical measurements, reduced graphene oxide was electrochemically deposited onto the electrode surface. Moreover, gold nanoparticles were introduced to enhance the conductivity of the PTB film and facilitate subsequent aptamer modification. Two common biological species, adenosine triphosphate (ATP) and cytochrome c (cyt c), were chosen as detection targets, and their corresponding aptasensors were successfully constructed and systematically evaluated. The proposed aptasensors based on the PTB-Au antifouling composite exhibited high sensitivity and specificity towards ATP and cyt c detection, and the detection limits were calculated to be 0.26 nM and 0.64 nM for ATP and cyt c, respectively. Hence, this work provides a simple approach to develop highly sensitive aptasensors without any labeling process, and thus promises its great application in biological analysis.


Assuntos
Aptâmeros de Nucleotídeos , Incrustação Biológica , Nanopartículas Metálicas , Trifosfato de Adenosina/análise , Proteínas Amiloidogênicas , Incrustação Biológica/prevenção & controle , Citocromos c , Ouro , Agregados Proteicos
20.
Elife ; 112022 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-35723573

RESUMO

Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.


Assuntos
Catarata , Cristalino , Catarata/metabolismo , Humanos , Inositol/análise , Inositol/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Agregados Proteicos
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