RESUMO
The amyloidoses are a group of protein misfolding diseases in which the precursor protein undergoes a conformational change that triggers the formation of amyloid fibrils in different tissues and organs, causing cell death and organ failure. Amyloidoses can be either localized or systemic. In localized amyloidosis, amyloid deposits form at the site of precursor protein synthesis, whereas in systemic amyloidosis, amyloid deposition occurs distant from the site of precursor protein secretion. We review the type of proteins and cells involved and what is known about the complex pathophysiology of these diseases. We focus on light chain amyloidosis to illustrate how biochemical and biophysical studies have led to a deeper understanding of the pathogenesis of this devastating disease. We also review current cellular, tissue, and animal models and discuss the challenges and opportunities for future studies of the systemic amyloidoses.
Assuntos
Amiloide/metabolismo , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Amiloidose/etiologia , Animais , Modelos Animais de Doenças , Humanos , Cadeias Leves de Imunoglobulina/química , Precursores de Proteínas/químicaRESUMO
Little is known about the mechanism of amyloid assembly in immunoglobulin light chain (AL) amyloidosis, in contrast to other amyloid diseases. Early events in the aggregation pathway are especially important, as these soluble species could be cytotoxic intermediates playing a critical role in the initiation of the amyloid assembly. In this work, we discuss the mechanism of the early events in in vitro fibril formation of immunoglobulin light chain AL-09 and AL-12 (involved in cardiac amyloidosis) and its germline (control) protein κI O18/O8. Previous work from our laboratory showed that AL-12 adopts a canonical dimer conformation (like the germline protein), whereas AL-09 presents an altered dimer interface as a result of somatic mutations. Both AL-12 and AL-09 aggregate with similar rates and significantly faster than the germline protein. AL-09 is the only protein in this study that forms stable oligomeric intermediates during the early stages of the aggregation reaction with some structural rearrangements that increase the thioflavin T fluorescence but maintain the same number of monomers in solution. The presence of the restorative mutation AL-09 H87Y changes the kinetics and the aggregation pathway compared to AL-09. The single restorative mutation AL-12 R65S slightly delayed the overall rate of aggregation as compared to AL-12. Collectively, our study provides a comprehensive analysis of species formed during amyloid nucleation in AL amyloidosis, shows a strong dependence between the altered dimer conformation and the formation of stable oligomeric intermediates, and sheds light on the structural features of amyloidogenic intermediates associated with cellular toxicity.
Assuntos
Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/metabolismo , Agregados Proteicos/fisiologia , Amiloidose/genética , Amiloidose/metabolismo , Humanos , Cadeias Leves de Imunoglobulina/genética , Mutação/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Light chain (AL) amyloidosis is a lethal disease characterized by the deposition of the immunoglobulin light chain into amyloid fibrils, resulting in organ dysfunction and failure. Amyloid fibrils have the ability to self-propagate, recruiting soluble protein into the fibril by a nucleation-polymerization mechanism, characteristic of autocatalytic reactions. Experimental data suggest the existence of a critical concentration for initiation of fibril formation. As such, the initial concentration of soluble amyloidogenic protein is expected to have a profound effect on the rate of fibril formation. In this work, we present in vitro evidence that fibril formation rates for AL light chains are affected by the protein concentration in a differential manner. De novo reactions of the proteins with the fastest amyloid kinetics (AL-09, AL-T05, and AL-103) do not present protein concentration dependence. Seeded reactions, however, exhibited weak protein concentration dependence. For AL-12, seeded and protein concentration dependence data suggest a synergistic effect for recruitment and elongation at low protein concentrations, while reactions of κI exhibited poor efficiency in nucleating and elongating preformed fibrils. Additionally, co-aggregation and cross seeding of κI variable domain (VL) and the κI full length (FL) light chain indicate that the presence of the constant domain in κI FL modulates fibril formation, facilitating the recruitment of κI VL. Together, these results indicate that the dominant process in fibril formation varies among the AL proteins tested with a differential dependence of the protein concentration.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cadeias Leves de Imunoglobulina/química , Agregados Proteicos , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Cristalografia por Raios X , Expressão Gênica , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Cinética , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND AIMS: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. METHODS: We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. RESULTS: We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. CONCLUSIONS: This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.
Assuntos
Amiloide/toxicidade , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/patologia , Amiloide/metabolismo , Apoptose , Células Cultivadas , Técnicas de Cocultura , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/terapia , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cadeias Leves de Imunoglobulina/química , Conformação Proteica , Amiloidose , Dicroísmo Circular , Humanos , Cinética , Estabilidade ProteicaRESUMO
Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.
Assuntos
Proteínas Amiloidogênicas/química , Sulfatos de Condroitina/química , Heparitina Sulfato/química , Cadeias kappa de Imunoglobulina/química , Mutação de Sentido Incorreto , Agregação Patológica de Proteínas , Substituição de Aminoácidos , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismoRESUMO
Clinical mutations in patients diagnosed with Type 2A von Willebrand disease (VWD) have been identified that break the single disulfide bond linking N- and C-termini in the vWF A1 domain. We have modeled the effect of these mutations on the disulfide-bonded structure of A1 by reducing and carboxy-amidating these cysteines. Solution biophysical studies show that loss of this disulfide bond induces a molten globule conformational state lacking global tertiary structure but retaining residual secondary structure. The conformational dependence of platelet adhesion to these native and molten globule states of A1 is quantitatively compared using real-time high-speed video microscopy analysis of platelet translocation dynamics under shear flow in a parallel plate microfluidic flow chamber. While normal platelets translocating on surface-captured native A1 domain retain the catch-bond character of pause times that increase as a function of shear rate at low shear and decrease as a function of shear rate at high shear, platelets that interact with A1 lacking the disulfide bond remain stably attached and do not translocate. Based on these findings, we propose that the shear stress-sensitive regulation of the A1-GPIb interaction is due to folding the tertiary structure of this domain. Removal of the tertiary structure by disrupting the disulfide bond destroys this regulatory mechanism resulting in high-strength interactions between platelets and vWF A1 that are dependent only on residual secondary structure elements present in the molten globule conformation.
Assuntos
Plaquetas/fisiologia , Reologia , Resistência ao Cisalhamento , Fator de von Willebrand/química , Acrilamida/metabolismo , Plaquetas/efeitos dos fármacos , Cromatografia em Gel , Dissulfetos/metabolismo , Guanidina/farmacologia , Humanos , Oxirredução/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Desnaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Reologia/efeitos dos fármacos , Resistência ao Cisalhamento/efeitos dos fármacos , Espectrometria de Fluorescência , Temperatura , Triptofano/metabolismoRESUMO
Light chain (AL) amyloidosis is a progressive, degenerative disease characterized by the misfolding and amyloid deposition of immunoglobulin light chain (LC). The amyloid deposits lead to organ failure and death. Our laboratory is specifically interested in cardiac involvement of AL amyloidosis. We have previously shown that the fibrillar aggregates of LC proteins can be cytotoxic and arrest the growth of human RFP-AC16 cardiomyocytes in vitro. We showed that adipose-derived mesenchymal stromal cells (AMSC) can rescue the cardiomyocytes from the fibril-induced growth arrest through contact-dependent mechanisms. In this study, we examined the transcriptome changes of human cardiomyocytes and AMSC in the presence of AL amyloid fibrils. The presence of fibrils causes a 'priming' immune response in AMSC associated with interferon associated genes. Exposure to AL fibrils induced changes in the pathways associated with immune response and extracellular matrix components in cardiomyocytes. We also observed upregulation of innate immune-associated transcripts (chemokines, cytokines, and complement), suggesting that amyloid fibrils initiate an innate immune response on these cells, possibly due to phenotypic transformation. This study corroborates and expands our previous studies and identifies potential new immunologic mechanisms of action for fibril toxicity on human cardiomyocytes and AMSC rescue effect on cardiomyocytes.
Assuntos
Biomarcadores/análise , Cadeias Leves de Imunoglobulina/imunologia , Amiloidose de Cadeia Leve de Imunoglobulina/imunologia , Inflamação/imunologia , Células-Tronco Mesenquimais/imunologia , Miócitos Cardíacos/imunologia , Apoptose , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Inflamação/metabolismo , Inflamação/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (â¼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.
RESUMO
We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.
Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Receptores Virais/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/uso terapêutico , Cricetinae , Microscopia Crioeletrônica , Evolução Molecular Direcionada/métodos , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Common biophysical techniques like absorption and fluorescence spectroscopy, microscopy, and light scattering studies have been in use to investigate fibril assembly for a long time. However, there is sometimes a lack of consensus from the findings of an individual technique when compared in parallel with the other techniques. In this chapter, we aim to provide a concise compilation of techniques that can effectively be used to obtain a comprehensive representation of the structural, aggregation, and toxicity determinants in immunoglobulin light chain amyloidosis. We start by giving a brief introduction on amyloid assembly and the advantages of using simple and readily available techniques to study aggregation. After an overview on preparation of protein to set up parallel experiments, we provide a systematic description of the in vitro techniques used to study aggregation in AL protein. Additionally, we thoroughly discuss the steps needed in our experience during the individual experiments for better reproducibility and data analysis.
Assuntos
Amiloide/química , Bioensaio , Cadeias Leves de Imunoglobulina/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Amiloidose/diagnóstico , Apoptose , Benzotiazóis/química , Benzotiazóis/metabolismo , Bioensaio/métodos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Difusão Dinâmica da Luz , Cadeias Leves de Imunoglobulina/metabolismo , Tamanho da Partícula , Espectrometria de FluorescênciaRESUMO
In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.
Assuntos
Entamoeba histolytica/enzimologia , Triose-Fosfato Isomerase/química , Animais , Dimerização , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , TermodinâmicaRESUMO
Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.
Assuntos
Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/fisiopatologia , Multimerização Proteica , Amiloide/química , Amiloide/genética , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Rearranjo Gênico , Glicosaminoglicanos/metabolismo , Humanos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Mutação , Plasmócitos/metabolismo , Estabilidade ProteicaRESUMO
Light chain (AL) amyloidosis is a systemic disease characterized by the formation of immunoglobulin light-chain fibrils in critical organs of the body. The light-chain protein AL-09 presents one severe case of cardiac AL amyloidosis, which contains seven mutations in the variable domain (VL) relative to its germline counterpart, κI O18/O8 VL. Three of these mutations are non-conservative-Y87H, N34I, and K42Q-and previous work has shown that they are responsible for significantly reducing the protein's thermodynamic stability, allowing fibril formation to occur with fast kinetics and across a wide-range of pH conditions. Currently, however, there is extremely limited structural information available which explicitly describes the residues that are involved in supporting the misfolded fibril structure. Here, we assign the site-specific 15N and 13C chemical shifts of the rigid residues of AL-09 VL fibrils by solid-state NMR, reporting on the regions of the protein involved in the fibril as well as the extent of secondary structure.
Assuntos
Amiloide/química , Cadeias Leves de Imunoglobulina/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Modelos Moleculares , Conformação Proteica em Folha beta , Domínios ProteicosRESUMO
Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.
Assuntos
Amiloide/química , Fotoquímica/métodos , Tiazóis/química , Animais , Benzotiazóis , HumanosRESUMO
It is now accepted that the ability of a protein to form amyloid fibrils could be associated both kinetic and thermodynamic protein folding parameters. A recent study from our laboratory using recombinant full-length (encompassing the variable and constant domain) immunoglobulin light chains found a strong kinetic control of the protein unfolding for these proteins. In this study, we are extending our analysis by using urine-derived Bence Jones proteins (BJPs) from five patients with light chain (AL) amyloidosis and four patients with multiple myeloma (MM). We observed lower stability in κ proteins compared to λ proteins (for both MM and AL proteins) in agreement with previous studies. The kinetic component of protein stability is not a universal feature of BJPs and the hysteresis observed during refolding reactions could be attributed to the inability of the protein to refold all domains. The most stable proteins exhibited 3-state unfolding transitions. While these proteins do not refold reversibly, partial refolding shows 2-state partial refolding transitions, suggesting that one of the domains (possibly the variable domain) does not refold completely. Sequences were aligned with their respective germlines and the location and nature of the mutations were analyzed. The location of the mutations were analyzed and compared with the stability and amyloidogenic properties for the proteins in this study, increasing our understanding of light chain unfolding and amyloidogenic potential.
Assuntos
Amiloidose/patologia , Proteína de Bence Jones/química , Mieloma Múltiplo/patologia , Sequência de Aminoácidos , Amiloidose/metabolismo , Proteína de Bence Jones/metabolismo , Proteína de Bence Jones/urina , Dicroísmo Circular , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina , Cinética , Mieloma Múltiplo/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Alinhamento de Sequência , TemperaturaRESUMO
Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (VL) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 VL. Previous work has demonstrated that the thermodynamic stability of native AL-09 VL is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 VL sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 VL forms an extensively ordered and ß-strand-rich fibril structure. Furthermore, we demonstrate that the predominant ß-sheet secondary structure and rigidity observed for in vitro prepared AL-09 VL fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils.
RESUMO
BACKGROUND: Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM. METHODS AND FINDINGS: We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains. CONCLUSION: The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.
Assuntos
Amiloide/imunologia , Proteínas Amiloidogênicas/imunologia , Amiloidose/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Mieloma Múltiplo/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Prognóstico , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Termodinâmica , Ureia/farmacologiaRESUMO
BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.
Assuntos
Colesterol/administração & dosagem , Endotélio Vascular/efeitos dos fármacos , Gangliosídeos/administração & dosagem , Amiloidose de Cadeia Leve de Imunoglobulina/complicações , Fosfatidilcolinas/administração & dosagem , Doenças Vasculares/prevenção & controle , Tecido Adiposo/irrigação sanguínea , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Sobrevivência Celular/fisiologia , Combinação de Medicamentos , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/prevenção & controle , Masculino , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , Nanopartículas/administração & dosagem , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Papaverina/farmacologia , Ácido Peroxinitroso/biossíntese , Interferência de RNA/fisiologia , RNA Interferente Pequeno/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Transfecção , Doenças Vasculares/fisiopatologia , Vasodilatadores/farmacologiaRESUMO
Light chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding-prone immunoglobulin light-chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL-12 protein by restoring four nonconservative mutations to their germline (wild-type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL-12 stability and fibril formation and restoring them to the wild-type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas-Mejia and Ramirez-Alvarado, Ann Rev Biochem 2013;82:745-774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL-12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.