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1.
Annu Rev Biochem ; 89: 1-19, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32343910

RESUMO

It is impossible to do justice in one review article to a researcher of the stature of Christopher Dobson. His career spanned almost five decades, resulting in more than 870 publications and a legacy that will continue to influence the lives of many for decades to come. In this review, I have attempted to capture Chris's major contributions: his early work, dedicated to understanding protein-folding mechanisms; his collaborative work with physicists to understand the process of protein aggregation; and finally, his later career in which he developed strategies to prevent misfolding. However, it is not only this body of work but also the man himself who inspired an entire generation of scientists through his patience, ability to mentor, and innate generosity. These qualities remain a hallmark of the way in which he conducted his research-research that will leave a lasting imprint on science.

2.
Annu Rev Biochem ; 89: 529-555, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32097570

RESUMO

Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.


Assuntos
Chaperonas Moleculares/genética , Técnicas de Sonda Molecular , Proteoma/genética , Deficiências na Proteostase/genética , Proteostase/genética , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Meia-Vida , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Chaperonas Moleculares/metabolismo , Agregados Proteicos , Engenharia de Proteínas/métodos , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteoma/química , Proteoma/metabolismo , Proteostase/efeitos dos fármacos , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
3.
Annu Rev Biochem ; 89: 389-415, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32569518

RESUMO

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.


Assuntos
Escherichia coli/genética , Chaperonas Moleculares/genética , Biossíntese de Proteínas , Proteoma/química , Ribossomos/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteoma/biossíntese , Proteoma/genética , Proteostase/genética , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Ribossomos/metabolismo , Ribossomos/ultraestrutura
4.
Cell ; 174(5): 1216-1228.e19, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30057111

RESUMO

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Peso Corporal , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Guanidinas/química , Células HeLa , Humanos , Doença de Huntington/metabolismo , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Proteostase , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de Superfície
5.
Annu Rev Biochem ; 86: 21-26, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28441058

RESUMO

The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia
6.
Mol Cell ; 84(8): 1512-1526.e9, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38508184

RESUMO

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.


Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de Proteína
7.
Annu Rev Cell Dev Biol ; 33: 439-465, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28992440

RESUMO

Cells and organisms have evolved numerous mechanisms to cope with and to adapt to unexpected challenges and harsh conditions. Proteins are essential to perform the vast majority of cellular and organismal functions. To maintain a healthy proteome, cells rely on a network of factors and pathways collectively known as protein quality control (PQC) systems, which not only ensure that newly synthesized proteins reach a functional conformation but also are essential for surveillance, prevention, and rescue of protein defects. The main players of PQC systems are chaperones and protein degradation systems: the ubiquitin-proteasome system and autophagy. Here we provide an integrated overview of the diverse PQC systems in eukaryotic cells in health and diseases, with an emphasis on the key regulatory aspects and their cross talks. We also highlight how PQC regulation may be exploited for potential therapeutic benefit.


Assuntos
Proteínas/metabolismo , Aminoácidos/metabolismo , Animais , Doença , Células Eucarióticas/metabolismo , Homeostase , Humanos , Estresse Fisiológico
8.
Annu Rev Biochem ; 84: 435-64, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25784053

RESUMO

Loss of protein homeostasis (proteostasis) is a common feature of aging and disease that is characterized by the appearance of nonnative protein aggregates in various tissues. Protein aggregation is routinely suppressed by the proteostasis network (PN), a collection of macromolecular machines that operate in diverse ways to maintain proteome integrity across subcellular compartments and between tissues to ensure a healthy life span. Here, we review the composition, function, and organizational properties of the PN in the context of individual cells and entire organisms and discuss the mechanisms by which disruption of the PN, and related stress response pathways, contributes to the initiation and progression of disease. We explore emerging evidence that disease susceptibility arises from early changes in the composition and activity of the PN and propose that a more complete understanding of the temporal and spatial properties of the PN will enhance our ability to develop effective treatments for protein conformational diseases.


Assuntos
Envelhecimento/patologia , Proteínas/metabolismo , Animais , Estresse do Retículo Endoplasmático , Humanos , Redes e Vias Metabólicas , Doenças Neurodegenerativas/patologia , Proteólise
9.
Mol Cell ; 82(8): 1477-1491, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35452616

RESUMO

Endoplasmic reticulum quality control (ERQC) pathways comprising chaperones, folding enzymes, and degradation factors ensure the fidelity of ER protein folding and trafficking to downstream secretory environments. However, multiple factors, including tissue-specific secretory proteomes, environmental and genetic insults, and organismal aging, challenge ERQC. Thus, a key question is: how do cells adapt ERQC to match the diverse, ever-changing demands encountered during normal physiology and in disease? The answer lies in the unfolded protein response (UPR), a signaling mechanism activated by ER stress. In mammals, the UPR comprises three signaling pathways regulated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Upon activation, these UPR pathways remodel ERQC to alleviate cellular stress and restore ER function. Here, we describe how UPR signaling pathways adapt ERQC, highlighting their importance for maintaining ER function across tissues and the potential for targeting the UPR to mitigate pathologies associated with protein misfolding diseases.


Assuntos
Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Animais , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Mamíferos , Controle de Qualidade , Transdução de Sinais
10.
Mol Cell ; 82(17): 3193-3208.e8, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35853451

RESUMO

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.


Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Chaperonas Moleculares/metabolismo
11.
Trends Biochem Sci ; 49(9): 761-774, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39168791

RESUMO

Loss of protein homeostasis (proteostasis) is a common hallmark of aging and age-associated diseases. Considered as the guardian of proteostasis, the proteostasis network (PN) acts to preserve the functionality of proteins during their lifetime. However, its activity declines with age, leading to disease manifestation. While reactive oxygen species (ROS) were traditionally considered culprits in this process, recent research challenges this view. While harmful at high concentrations, moderate ROS levels protect the cell against age-mediated onset of proteotoxicity by activating molecular chaperones, stress response pathways, and autophagy. This review explores the nuanced roles of ROS in proteostasis and discusses the most recent findings regarding the redox regulation of the PN and its potential in extending healthspan and delaying age-related pathologies.


Assuntos
Proteostase , Espécies Reativas de Oxigênio , Humanos , Espécies Reativas de Oxigênio/metabolismo , Animais , Oxidantes/metabolismo , Autofagia , Oxirredução , Envelhecimento/metabolismo , Estresse Oxidativo
12.
Proc Natl Acad Sci U S A ; 121(15): e2320456121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38568974

RESUMO

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.


Assuntos
Príons , Tauopatias , Humanos , Proteínas tau/metabolismo , Tauopatias/metabolismo , Isoformas de Proteínas/metabolismo , Príons/metabolismo , Peptídeos , Aminoácidos
13.
Proc Natl Acad Sci U S A ; 121(34): e2315005121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39133858

RESUMO

The process of protein phase separation into liquid condensates has been implicated in the formation of membraneless organelles (MLOs), which selectively concentrate biomolecules to perform essential cellular functions. Although the importance of this process in health and disease is increasingly recognized, the experimental identification of proteins forming MLOs remains a complex challenge. In this study, we addressed this problem by harnessing the power of AlphaFold2 to perform computational predictions of the conformational properties of proteins from their amino acid sequences. We thus developed the CoDropleT (co-condensation into droplet transformer) method of predicting the propensity of co-condensation of protein pairs. The method was trained by combining experimental datasets of co-condensing proteins from the CD-CODE database with curated negative datasets of non-co-condensing proteins. To illustrate the performance of the method, we applied it to estimate the propensity of proteins to co-condense into MLOs. Our results suggest that CoDropleT could facilitate functional and therapeutic studies on protein condensation by predicting the composition of protein condensates.


Assuntos
Proteínas , Proteínas/química , Proteínas/metabolismo , Biologia Computacional/métodos , Organelas/metabolismo , Conformação Proteica , Bases de Dados de Proteínas , Sequência de Aminoácidos
14.
Semin Cell Dev Biol ; 156: 107-120, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-37734998

RESUMO

The ubiquitin proteasome system maintains protein homeostasis by regulating the breakdown of misfolded proteins, thereby preventing misfolded protein aggregates. The efficient elimination is vital for preventing damage to the cell by misfolded proteins, known as proteotoxic stress. Proteotoxic stress can lead to the collapse of protein homeostasis and can alter the function of the ubiquitin proteasome system. Conversely, impairment of the ubiquitin proteasome system can also cause proteotoxic stress and disrupt protein homeostasis. This review examines two impacts of proteotoxic stress, 1) disruptions to ubiquitin homeostasis (ubiquitin stress) and 2) disruptions to proteasome homeostasis (proteasome stress). Here, we provide a mechanistic description of the relationship between proteotoxic stress and the ubiquitin proteasome system. This relationship is illustrated by findings from several protein misfolding diseases, mainly neurodegenerative diseases, as well as from basic biology discoveries from yeast to mammals. In addition, we explore the importance of the ubiquitin proteasome system in endoplasmic reticulum quality control, and how proteotoxic stress at this organelle is alleviated. Finally, we highlight how cells utilize the ubiquitin proteasome system to adapt to proteotoxic stress and how the ubiquitin proteasome system can be genetically and pharmacologically manipulated to maintain protein homeostasis.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Animais , Ubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse Proteotóxico , Proteínas/metabolismo , Mamíferos/metabolismo
15.
Mol Cell ; 70(6): 1025-1037.e5, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29861160

RESUMO

When faced with proteotoxic stress, cells mount adaptive responses to eliminate aberrant proteins. Adaptive responses increase the expression of protein folding and degradation factors to enhance the cellular quality control machinery. However, it is unclear whether and how this augmented machinery acquires new activities during stress. Here, we uncover a regulatory cascade in budding yeast that consists of the hydrophilin protein Roq1/Yjl144w, the HtrA-type protease Ynm3/Nma111, and the ubiquitin ligase Ubr1. Various stresses stimulate ROQ1 transcription. The Roq1 protein is cleaved by Ynm3. Cleaved Roq1 interacts with Ubr1, transforming its substrate specificity. Altered substrate recognition by Ubr1 accelerates proteasomal degradation of misfolded as well as native proteins at the endoplasmic reticulum membrane and in the cytosol. We term this pathway stress-induced homeostatically regulated protein degradation (SHRED) and propose that it promotes physiological adaptation by reprogramming a key component of the quality control machinery.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , Saccharomyces cerevisiae/enzimologia , Serina Endopeptidases/metabolismo , Estresse Fisiológico/fisiologia , Especificidade por Substrato , Ubiquitina/metabolismo
16.
J Biol Chem ; 300(6): 107315, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38663827

RESUMO

Lewy bodies (LB) are aberrant protein accumulations observed in the brain cells of individuals affected by Parkinson's disease (PD). A comprehensive analysis of LB proteome identified over a hundred proteins, many co-enriched with α-synuclein, a major constituent of LB. Within this context, OTUB1, a deubiquitinase detected in LB, exhibits amyloidogenic properties, yet the mechanisms underlying its aggregation remain elusive. In this study, we identify two critical sites in OTUB1-namely, positions 133 and 173-that significantly impact its amyloid aggregation. Substituting alanine at position 133 and lysine at position 173 enhances both thermodynamic and kinetic stability, effectively preventing amyloid aggregation. Remarkably, lysine at position 173 demonstrates the highest stability without compromising enzymatic activity. The increased stability and inhibition of amyloid aggregation are attributed mainly to the changes in the specific microenvironment at the hotspot. In our exploration of the in-vivo co-occurrence of α-synuclein and OTUB1 in LB, we observed a synergistic modulation of each other's aggregation. Collectively, our study unveils the molecular determinants influencing OTUB1 aggregation, shedding light on the role of specific residues in modulating aggregation kinetics and structural transition. These findings contribute valuable insights into the complex interplay of amino acid properties and protein aggregation, with potential implications for understanding broader aspects of protein folding and aggregation phenomena.


Assuntos
alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/química , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/química , Agregados Proteicos , Corpos de Lewy/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Amiloide/metabolismo , Amiloide/química , Estabilidade Proteica , Estabilidade Enzimática , Cinética
17.
J Biol Chem ; 300(5): 107231, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38537700

RESUMO

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.


Assuntos
Neuropatias Amiloides , Peptídeos e Proteínas de Sinalização Intercelular , Rim , Fluxo Plasmático Renal , Humanos , Amiloide/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Rim/irrigação sanguínea , Rim/fisiopatologia , Estresse Mecânico , Neuropatias Amiloides/metabolismo , Neuropatias Amiloides/fisiopatologia , Resistência ao Cisalhamento , Agregados Proteicos
18.
J Biol Chem ; 300(8): 107507, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38944121

RESUMO

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We used phage display technology to identify novel single-chain variable fragments that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size-exclusion chromatography combined with multiangle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.


Assuntos
Epitopos , Gelsolina , Humanos , Gelsolina/química , Gelsolina/metabolismo , Gelsolina/imunologia , Epitopos/imunologia , Epitopos/química , Amiloidose/metabolismo , Amiloidose/imunologia , Amiloide/metabolismo , Amiloide/imunologia , Agregados Proteicos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Agregação Patológica de Proteínas/metabolismo
19.
FASEB J ; 38(14): e23843, 2024 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-39072789

RESUMO

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.


Assuntos
Proteínas PrPSc , Dobramento de Proteína , Animais , Dobramento de Proteína/efeitos dos fármacos , Proteínas PrPSc/metabolismo , Proteínas PrPSc/química , Camundongos , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Príons/metabolismo
20.
Cell Mol Life Sci ; 81(1): 271, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38888668

RESUMO

Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin-proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Ubiquitinação , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Retículo Endoplasmático/metabolismo , Animais , Mutação , Ubiquitina/metabolismo
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