Folding Disorders Research Articles

A bibliometric analysis of light chain amyloidosis from 2005 to 2024: research trends and hot spots.

Light chain (AL) amyloidosis stands as the most prevalent subtype of systemic amyloidosis, encompassing a group of rare diseases. Here, we evaluated the scientific landscape of AL amyloidosis to investigate research trends and identify hotspots within the field. Relevant studies on AL amyloidosis published over the past two decades were retrieved from the Web of Science Core Collection. The publications between 2005 and 2024 were subjected to bibliometric analyses, leveraging tools including CiteSpace, VOSviewer, RStudio and MS Excel to analyse and visualize the annual publication trend, co-occurrence patterns, collaborative networks among countries, organizations, and authors. Burst keywords and references were also examined to obtain the research history, and emerging hotspots. The bibliometric analysis included 2,864 articles published between 2005 and 2024. The most productive journal is Amyloid-Journal of Protein Folding Disorders. The United States, along with several developed nations, emerges as a dominant force in international AL amyloidosis research. "AL amyloidosis" and "cardiac amyloidosis" were the primary hotspots over the past two decades, and "Biomarkers," "Cardiac amyloidosis," and "treatment" would be future trends. This bibliometric analysis examined the research developments in AL amyloidosis over the past two decades using bibliometric software. Recent research in this field primarily focuses on two main areas: clinical diagnosis and treatment of AL amyloidosis, as well as cardiac amyloidosis. Emphasis is placed on understanding the mechanisms underlying immunoglobulin light chain aggregation and deposition to mitigate organ involvement.

Human in vitro disease modeling of ATTR cardiac amyloidosis

Abstract Background Amyloidosis is a systemic protein-folding disorder characterized by extracellular misfolded fibrillar protein aggregates that are insoluble and resistant to proteolysis, leading to progressive and vital organ damage [1]. Transthyretin amyloidosis (ATTR) is one of the primary amyloidosis types that affect the heart. Based on the TTR gene sequence, ATTR is further classified into wild-type ATTR (ATTRwt) and hereditary ATTR (ATTRv). Advanced stage TTR amyloid infiltration of the myocardium results in progressive thickening and stiffening of the ventricle wall, and symptoms of restrictive cardiomyopathy and congestive heart failure. Due to the lack of specificity of cardiac amyloidosis symptoms, misdiagnosis and delays in diagnosis are common, resulting in worsening symptoms, organ damage, and increased mortality [2]. Despite the development of several disease-modifying agents [3], current experimental cell and animal models do not adequately represent the human situation, thereby limiting disease understanding and therapeutic discovery. Purpose This project aims to develop an in vitro ATTR disease model, which is critical to define early disease stages and disease progression, map human disease-specific pathways, and screen for novel drugs or validating cutting-edge therapeutic strategies. Methods Recombinant WT-TTR and V122I mutated TTR (V122I) were prepared using E.coli protein production system. TTR purification and acid-mediated TTR-fibril were characterized by SDS-PAGE, Thioflavin T fluorescence measurement, and transmission electron microscope (EM). iPSC-derived cardiomyocytes (iPS-CMs) were differentiated using GiWi protocol as described [4,5]. iPS-CMs or HUVECs were seeded on TTR or TTR-fibril, and we measured beating rate, cell viability (AlamarBlue and LiveDead), and cell/fibril morphology using immunofluorescence. Results Purified recombinant WT and V122I TTR was successfully generated (Figure 1A). Fluorescence increase (Thioflavin T) and EM revealed the presence of TTR-fibril (Figure 1B). iPS-CMs seeded on TTR-fibril resulted in a dose- and time-dependent increase in cell death. Confocal microscopy revealed the attachment of TTR-fibril on iPS-CMs, leading to sarcomere disarray, loss of cell-to-cell coupling, resulting in irregular beating (Figure 2A). Additionally, we found drastically affected viability of HUVECs upon TTR-fibril exposure (Figure 2B). Conclusion Here, we demonstrate an established method to generate purified TTR and TTR-fibril. TTR-fibril attached to the surface of iPS-CMs decreased cell viability and functionality. Similar reduction in HUVEC viability were observed as well. These findings reflect the cytotoxicity of TTR-fibril in a human in vitro model.TTR and TTR-fibril characterizationCell viability up TTR-fibril exposure

Enhanced Inhibition of Amyloid Formation by Heat Shock Protein 90 Immobilized on Nanoparticles.

As the population ages, an epidemic of neurodegenerative diseases with devastating social consequences is looming. To address the pathologies leading to amyloid-related dementia, novel therapeutic strategies must be developed for the treatment or prevention of neural protein-folding disorders. Nanotechnology will be crucial to this scenario, especially in the design of nanoscale systems carrying therapeutic compounds that can navigate the nervous system and identify amyloid to treat it in situ. In this line, we have recently designed a highly simplified and versatile nanorobot consisting of a protein coating based on the heat shock protein 90 (Hsp90) chaperone that not only propels nanoparticles using ATP but also endows them with the extraordinary ability to fold and restore the activity of heat-denatured proteins. Here, we assess the effectiveness of these nanosystems in inhibiting/reducing the aggregation of amyloidogenic proteins. Using Raman spectroscopy, we qualitatively and quantitatively analyze amyloid by identifying and semi-quantifying the Amide I band. Our findings indicate that the coupling of Hsp90 to nanoparticles results in a more potent inhibition of amyloid formation when compared to the soluble protein. We propose that this enhanced performance may be attributed to enhanced release-capture cycles of amyloid precursor oligomers by Hsp90 molecules nearby on the nanosurface. Intelligent biocompatible coatings, like the one described here, that enhance the diffusivity and self-propulsion of nanoparticles while enabling them to carry out critical functions such as environmental scanning, identification, and amyloid prevention, present an exceptional opportunity for the development of advanced nanodevices in biomedical applications. This approach, which combined active biomolecules with synthetic materials, is poised to reveal remarkable prospects in the field of nanomedicine and biotechnology.

The Clusterin/Von Willebrand Factor Ratio Is Significantly Lower in Marrow Plasma from AL λ-Type Than from λ-Isotype Monoclonal Gammopathy Patients

Background: Our primary objective is to develop methods that enable early diagnosis of AL or risk of AL during the prodromal decade prior to symptomatic presentation (Weiss et al. J Clin Oncol 2014;32:2699). There are parameters that increase risk of AL such as the presence of a monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM) or having AL-related IGVL genes encoding light-chain production in clonal plasma cells (Zhou et al. J Clin Oncol 2019;37:8010; Kourelis et al. Blood 2017;129:299; Kyle et al. N Engl J Med 2007;356:2582). Previous studies have shown that clusterin (apolipoprotein J) may play a variety of roles in protein folding disorders, possibly binding and clearing misfolded proteins. Like apolipoprotein E (apoE), clusterin is found in amyloid deposits of all types including the arterial deposits of cerebral amyloid angiopathy; moreover, it interacts cooperatively with apoE in Alzheimer's (Endo et al. Acta Neuropathol Commun 2019;7:12; DeMattos et al. Neuron 2004;41:193). Furthermore, serum levels of clusterin are reduced in patients with AL cardiac involvement (Filippini et al. Glia2021;69:681; Greene et al. Biochemistry 2015;54:268; Greene et al. Am J Pathol2011;178:61; Lee et al. Biochem Biophys Res Commun2009;388:256). A previous study has also shown that von Willebrand factor (vWF) levels are significantly elevated in AL patients, possibly due to endothelial cell dysfunction (Kastritis et al. Blood 2016;128:405); AL amyloidosis commonly involves the vasculature. Seeking additional parameters for likelihood of AL we asked whether the ratio of clusterin to vWF (C/v) in marrow plasma differed among AL, MGUS and SMM patients. Materials & Methods: λ-isotype MGUS or SMM patients from multiple sites in the USA with a difference between involved and uninvolved FLC > 23mg/L and a κ-to-λ ratio below normal and no evidence of amyloid on prior studies consented to participate in a clinical study screening for AL (NCT04615572) and had bone marrow aspirates sent overnite to Tufts. In addition, newly diagnosed AL patients consented to have blood and marrow aspirates obtained for research on an IRB-approved study. All samples were heparinized and had plasma separated initially and aliquoted for storage at -20o C. We also obtained peripheral blood plasma from normal individuals, ages unknown (Precision for Medicine; Norton, MA). Clusterin and vWF ELISA were performed on thawed plasma following manufacturers’ instructions (R&D Systems; Minneapolis, MN). GraphPad PRISM V5 was used for statistical analyses. Results: Twelve MGUS and 16 SMM patients, all λ-isotype, enrolled on NCT04615572, met FLC criteria and had marrow shipped to Tufts; 2 of the SMM patients were found to have AL and are included with 24 AL λ-type patients whose marrow aspirates were obtained in the same time period. There were no differences in age or gender, but the percentage of marrow plasma cells in SMM and AL patients were significantly greater than in MGUS patients. Comparisons of AL, SMM and MGUS iFLC λ levels did not achieve statistical significance. Medians were 338, 182 and 346 mg/L respectively. Marrow plasma from AL patients had significantly lower C/v ratios than those in either MGUS or SMM specimens (Figure 1). There was no correlation between λ FLC and the C/v ratio (r = -0.20, P > 0.05). Compared to normal individuals (n=20) with a median C/v ratio of 16.9x104 (range, 2.7-56.5), the C/v ratio in peripheral blood in newly diagnosed AL patients (n=15) was significantly lower at a median of 1.5x104 (0.8-2.0) (P<0.01, Mann-Whitney two-tailed). Conclusion: These preliminary data suggest that the C/v ratio is a potential parameter of interest in the construction of a likelihood algorithm for AL in patients with monoclonal gammopathies. The rationale for the ratio is that with progression during the prodromal period prior to significant organ damage clusterin may decrease while vWF may increase. Whether peripheral blood may provide a useful source of plasma is unclear and whether ratios below a threshold or changes in the ratio over time may signal risk of AL or be significant remain questions for further exploration. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IgM-Related Immunoglobulin Light Chain (AL) Amyloidosis

Waldenström macroglobulinemia (WM) is a rare lymphoplasmacytic disorder characterized by an IgM paraprotein. The clinical presentation of WM varies and can include common manifestations such as anemia and hyperviscosity, in addition to less common features such as cryoglobulinemia, IgM-related neuropathy, and immunoglobulin light chain (AL) amyloidosis. Amyloidosis is a protein-folding disorder in which vital organ damage occurs due to the accumulation of misfolded protein aggregates. The most common type of amyloidosis in patients with an IgM paraprotein is AL amyloidosis, although other types of amyloidosis may occur. IgM-related amyloidosis has distinct clinical features when compared with other subtypes of AL amyloidosis. This review highlights the diagnostic criteria of IgM-related AL amyloidosis, as well as the clinical characteristics and treatment options for this disorder.

Structural Determinant of β-Amyloid Formation: From Transmembrane Protein Dimerization to β-Amyloid Aggregates.

Most neurodegenerative diseases have the characteristics of protein folding disorders, i.e., they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer's disease is one of these diseases. It is characterized by two types of lesions: neurofibrillary tangles (NFTs) composed of tau proteins and senile plaques, formed essentially of amyloid peptides (Aβ). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aβ deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aβ production, and to understand the processes leading to the formation of different Aβ aggregates, e.g., fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aβ production essentially by regulating membrane protein dimerization, and subsequently Aβ aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept is critical to better decipher the patterns switching APP protein conformation from physiological to pathological and improve our understanding of the mechanisms underpinning the formation of amyloid fibrils that devastate neuronal functions.

The Extracellular Chaperone Clusterin in Aβ and Non‐Aβ Cerebral Amyloidoses

Clusterin, a multifunctional glycoprotein also known as apolipoprotein J, has been implicated in several physiological processes and is an important contributor to many pathological conditions, including Alzheimer’s disease (AD) in which genetic variants and plasma levels of the apolipoprotein were identified as risk factors for the disorder. To investigate whether clusterin exerted a more general role in the pathophysiology of cerebral amyloidoses, we turned to two unrelated hereditary conditions, familial British and Danish dementias (FBD and FDD), which share striking neuropathological similarities with AD, including neurofibrillary degeneration in limbic areas as well as parenchymal and vascular amyloid deposition. Two different 4 kDa molecules – ABri in FBD and ADan in FDD – totally unrelated to the 4 kDa Alzheimer’s Aβ, are the main constituents of the amyloid deposits in these forms of cerebral amyloidoses. Mirroring findings in AD, a panel of immunohistochemical studies demonstrated clusterin co‐localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri‐ and ADan‐binding plasma protein. Solid‐phase enzyme‐linked immunosorbent assays highlighted a specific saturable binding of clusterin to ABri and ADan. Non‐linear regression analysis of the data revealed Kd values within the same low nanomolar range as those demonstrated for the clusterin‐Aβ interaction. Consistent with its reported chaperone activity, clusterin showed a modulatory effect on ABri, ADan, and Aβ aggregation/fibrillization properties evaluated using thioflavin T, a dye that displays enhanced fluorescence upon binding fibrillar and protofibrillar amyloid conformations. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of protein folding disorders, including AD, FBD, and FDD – suggests the likelihood of a more complex role and a translational potential of clusterin in the amelioration/prevention of these pathogenic mechanisms.

A small molecule high throughput screening platform to profile conformational properties of nascent, ribosome-bound proteins

Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis. Transient de-novo folding intermediates therefore represent potential drug targets for pharmacological correction of protein folding disorders. Here we develop a FRET-based high-throughput screening (HTS) assay in 1,536-well format capable of identifying small molecules that interact with nascent polypeptides and correct genetic, cotranslational folding defects. Ribosome nascent chain complexes (RNCs) containing donor and acceptor fluorophores were isolated from cell free translation reactions, immobilized on Nickel-NTA/IDA beads, and imaged by high-content microscopy. Quantitative FRET measurements obtained from as little as 0.4 attomole of protein/bead enabled rapid assessment of conformational changes with a high degree of reproducibility. Using this assay, we performed a pilot screen of ~ 50,000 small molecules to identify compounds that interact with RNCs containing the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) harboring a disease-causing mutation (A455E). Screen results yielded 133 primary hits and 1 validated hit that normalized FRET values of the mutant nascent peptide. This system provides a scalable, tractable, structure-based discovery platform for screening small molecules that bind to or impact the folding of protein substrates that are not amenable to traditional biochemical analyses.

Healthcare Resource Utilization and Costs of Patients with AL Amyloidosis: An Analysis of Hospitalizations in the Premier Database

Healthcare Resource Utilization and Costs of Patients with AL Amyloidosis: An Analysis of Hospitalizations in the Premier Database

Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of these protein folding disorders – suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.

Bioreactors for Vocal Fold Tissue Engineering.

It is estimated that almost one-third of the United States population will be affected by a vocal fold (VF) disorder during their lifespan. Promising therapies to treat VF injury and scarring are mostly centered on VF tissue engineering strategies such as the injection of engineered biomaterials and cell therapy. VF tissue engineering, however, is a challenging field as the biomechanical properties, structure, and composition of the VF tissue change upon exposure to mechanical stimulation. As a result, the development of long-term VF treatment strategies relies on the characterization of engineered tissues under a controlled mechanical environment. In this review, we highlight the importance of bioreactors as a powerful tool for VF tissue engineering with a focus on the current state of the art of bioreactors designed to mimic phonation in vitro. We discuss the influence of the phonatory environment on the development, function, injury, and healing of the VF tissue and its importance for the development of efficient therapeutic strategies. A concise and comprehensive overview of bioreactor designs, principles, operating parameters, and scalability are presented. An in-depth analysis of VF bioreactor data to date reveals that mechanical stimulation significantly influences cell viability and the expression of proinflammatory and profibrotic genes in vitro. Although the precision and accuracy of bioreactors contribute to generating reliable results, diverse gene expression profiles across the literature suggest that future efforts should focus on the standardization of bioreactor parameters to enable direct comparisons between studies. Impact statement We present a comprehensive review of bioreactors for vocal fold (VF) tissue engineering with a focus on the influence of the phonatory environment on the development, function, injury, and healing of the VFs and the importance of mimicking phonation on engineered VF tissues in vitro. Furthermore, we put forward a strong argument for the continued development of bioreactors in this area with an emphasis on the standardization of bioreactor designs, principles, operating parameters, and oscillatory regimes to enable comparisons between studies.

Cutaneous insulin-derived amyloidosis presenting as hyperkeratotic nodules.

Amyloidosis consists of approximately 30 protein-folding disorders in which a specific soluble precursor protein aggregates to form the insoluble fibrils of amyloid. Insulin is the hypothesized precursor protein in localized cutaneous insulin-derived (AIns) amyloidosis. Amyloid deposition at insulin injection sites can interfere with absorption, leading to poor glucose control. Despite the increasing prevalence of diabetes mellitus and insulin use, there is a paucity of published cases of AIns amyloidosis and a lack of awareness of this condition among both dermatologists and general practitioners. We report 2 patients with insulin-dependent diabetes who developed hyperkeratotic nodules at insulin injection sites. Our cases complement the current data on iatrogenic amyloidosis and provide insight into this likely underreported phenomenon.

Patterns of coronary vascular involvement in patients with heart failure due to cardiac amyloidosis

Abstract Background Amyloidosis is a group of protein-folding disorders characterised by organ deposits, derived from one of several amyloidogenic precursor proteins. The involvement of coronary arteries has been previously described in amyloidosis, however the pattern of the disease in the coronaries is still unknown. Purpose The aim of this study is to characterise the pattern and severity of coronary artery lesions in cardiac amyloidosis. Methods We retrospectively compared patients with heart failure who tested positive (i.e. biopsy or gene tests – HF/CA+) against those that tested negative (HF/CA−) for cardiac amyloidosis. Groups were compared demographically and angiographically for qualitative and quantitative variables to determine patterns of involvement in the major epicardial coronary vessels. Results In total, 110 heart failure patients were included in the study, of whom 55 were HF/CA+ patients (88 lesions) and 55 were HF/CA− patients (66 lesions). Despite the advanced age in the HF/CA+, (74.53±11.02 vs 54.11±15.08; p=0.05), severe calcification was reported in HF/CA− group (4.5% vs to 0.0%; p=0.018). The HF/CA+ group also had fewer ostial lesions (3.4% vs. 15.15%, p=0.0095), reduced TIMI flow grade (83% vs 76%; p=0.21) and a higher TIMI frame count (30±12 Vs 27±11 frames; p=0.06). In the HF/CA+ group, compared to women, men had a significant number of tandem lesions (14.55% vs 0.0%, p=0.02). Men trended to have more ulcerations in comparison to women (9.09% vs 0.0%; p=0.15). Conclusion Overall, patients with HF/CA+ were older but found to have lesser calcified lesions, ostial involvement and a reduced anterograde blood flow. This is the first report outlining the coronary lesions in patients with HF/CA+. Funding Acknowledgement Type of funding source: None

Association of the DNASE1L3 rs35677470 polymorphism with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis: Structural biological insights.

Although genome-wide association studies (GWAS) have identified hundreds of autoimmune disease-associated loci, much of the genetics underlying these diseases remains unknown. In an attempt to identify potential causal variants, previous studies have determined that the rs35677470 missense variant of the Deoxyribonuclease I-like 3 (DNASE1L3) gene was associated with the development of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc). DNase1L3 is a member of the human DNase I family, representing a nuclease that cleaves double-stranded DNA during apoptosis and serving a role in the development of autoimmune diseases. The present study aimed to determine the role of the rs35677470 variant at the DNASE1L3 gene leading to the R206C mutation in SLE, RA and SSc. The underlying mechanism potentially affecting protein structure loss of function was also assessed. DNASE1L3 evolution was investigated to define conservation elements in the protein sequence. Additionally, 3D homology modeling and in silico mutagenesis was performed to localize the polymorphism under investigation. Evolutionary analysis revealed heavily conserved sequence elements among species, indicating structural/functional importance. In silico mutagenesis and 3D protein structural analysis also demonstrated the potentially varied impact of the DNASE1L3 (rs35677470) single nucleotide polymorphism (SNP), providing an explanation for its effect on the R206C variant. Structural analysis demonstrated that the rs35677470 SNP encodes a non-conservative amino acid variation, R206C, which disrupted the conserved electrostatic network holding secondary protein structure elements in position. Specifically, the R206 to E170 interaction forming part of a salt bridge network stabilizing two α-helices was interrupted, thereby affecting the molecular architecture. Previous studies on the effect of this SNP in Caucasian populations demonstrated lower DNAse1L3 activity levels, which is consistent with the current results. The present study comprehensively evaluated the shared autoimmune locus of DNASE1L3 (rs35677470), which produced an inactive form of DNaseIL3. Furthermore, structural analysis explained the potential role of the produced mutation by modifying the placement of structural elements and consequently introducing disorder in protein folding, affecting biological function.

TTRMDB: A database for structural and functional analysis on the impact of SNPs over transthyretin (TTR) using bioinformatic tools

Hereditary Transthyretin-associated amyloidosis (ATTR) is an autosomal dominant protein-folding disorder with adult-onset caused by mutation of transthyretin (TTR). TTR is characterized by extracellular deposition of amyloid, leading to loss of autonomy and finally, death. More than 100 distinct mutations in TTR gene have been reported from variable age of onset, clinical expression and penetrance data. Besides, the cure for the disease remains still obscure. Further, the prioritizing of mutations concerning the characteristic features governing the stability and pathogenicity of TTR mutant proteins remains unanswered, to date and thus, a complex state of study for researchers. Herein, we provide a full report encompassing the effects of every reported mutant model of TTR protein about the stability, functionality and pathogenicity using various computational tools. In addition, the results obtained from our study were used to create TTRMDB (Transthyretin mutant database), which could be easy access to researchers at http://vit.ac.in/ttrmdb.

Binding-induced folding under unfolding conditions: Switching between induced fit and conformational selection mechanisms

The chemistry of protein-ligand binding is the basis of virtually every biological process. Ligand binding can be essential for a protein to function in the cell by stabilizing or altering the conformation of a protein, particularly for partially or completely unstructured proteins. However, the mechanisms by which ligand binding impacts disordered proteins or influences the role of disorder in protein folding is not clear. To gain insight into this question, the mechanism of folding induced by the binding of a Pro-rich peptide ligand to the SH3 domain of phosphatidylinositol 3-kinase unfolded in the presence of urea has been studied using kinetic methods. Under strongly denaturing conditions, folding was found to follow a conformational selection (CS) mechanism. However, under mildly denaturing conditions, a ligand concentration-dependent switch in the mechanism was observed. The folding mechanism switched from being predominantly a CS mechanism at low ligand concentrations to being predominantly an induced fit (IF) mechanism at high ligand concentrations. The switch in the mechanism manifests itself as an increase in the reaction flux along the IF pathway at high ligand concentrations. The results indicate that, in the case of intrinsically disordered proteins too, the folding mechanism is determined by the concentration of the ligand that induces structure formation.

Dietary restriction improves proteostasis and increases life span through endoplasmic reticulum hormesis

Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPRER to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. Here, we show that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance. We found that only the IRE-1-XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1-dependent manner. We show that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Finally, we show that ER hormesis improves proteostasis and viability in a mammalian cellular model of neurodegenerative disease. Together, our study identifies a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.

Prevalence and prognostic value of D-dimer elevation in patients with AL amyloidosis.

Light chain (AL) amyloidosis is a protein folding disorder that can affect many different organ systems, in addition to the coagulation pathway. D-dimer, a measurement of fibrin degradation, is commonly elevated in hematologic malignancies, but the prevalence and significance of D-dimer elevation in AL amyloidosis is unknown. We conducted an analysis of 921 patients with AL amyloidosis that presented to the Boston University Amyloidosis Center. Baseline characteristics and laboratory data of the 897 patients included in the final cohort were analyzed. Four hundred twenty three patients (47%) had an elevated D-dimer (>0.5 μg/mL). Multivariate analysis demonstrated that a normal D-dimer level of ≤0.5 μg/mL, and a level of >0.5 μg/mL but <1 μg/mL, conferred a lower risk of mortality (HR 0.49 and 0.59, respectively) when compared to a D-dimer level ≥ 1 μg/mL. The increased risk of mortality in patients with a D-dimer level ≥ 1 μg/mL was present in all cardiac stages. The median overall survival based on D-dimer range of ≤0.5, >0.5 but <1, and ≥ 1 μg/mL was 5.86, 4.04, and 2.08 years, respectively (P < .001). This retrospective analysis demonstrates the high prevalence of D-dimer elevation in AL amyloidosis and confirms that this laboratory finding is independently associated with decreased survival.

SUN-187 LYSOZYME AMYLOIDOSIS – TWO NOVEL CASES WITH NEWLY DESCRIBED GENETIC MUTATIONS

Amyloidosis is a disorder of protein folding in which proteins are deposited as abnormal insoluble fibrils which disrupt tissue structures and cause disease. Lysozyme is a bacteriolytic enzyme present in secretions synthesised by gastrointestinal cells, macrophages and hepatocytes. Lysozyme amyloidosis is a rare form of hereditary amyloidosis which typically manifests with renal impairment, gastrointestinal symptoms and sicca syndrome. The natural history of lysozyme amyloidosis is felt to be indolent with slow progression of end organ damage associated with the condition. The complexity of diagnosing hereditary forms of amyloidosis likely results in underdiagnosis of the condition or inappropriate labeling of the condition as immunoglobulin associated amyloidosis. Measures utilised to identify immunoglobulin associated amyloidosis, such as serum or urinary protein measurements, are unable to utilised in identifying lysozyme amyloidosis with immunohistochemistry and molecular genetic testing being required for formal identification. We report two cases of patients with lysozyme amyloidosis managed by our service. Case 1 was referred to our service in May 2017. The 44-year-old woman was referred with chronic renal impairment and mild proteinuria (protein/creatinine ratio 22 mg/mmol) on a background of peptic ulcer disease managed with an oral proton pump inhibitor, chronic back pain, cigarette smoking, menorrhagia, dry eyes and bilateral knee osteoarthritis. Screening testing of serum free light chains, paraprotein, antinuclear antibody, complements and renal tract imaging were unremarkable. Renal biopsy identified heavy congo red deposits within glomerular, vascular and interstitial compartments. Transthyretin and amyloid A staining were negative. Further tissue assessment with mass spectrometry was performed identifying high levels of lysozyme consistent with lysozyme amyloidosis. Molecular genetic testing identified heterozygous presence for the c.176G>C variant in the LYZ gene, a variant of uncertain clinical significance. Case 2 was referred to our service in June 2018. The 43-year-old man presented with a creatinine of 251 with no background medical history. Screening testing of serum free light chains, paraprotein, antinuclear antibody, complements and renal tract imaging were unremarkable. Renal biopsy demonstrated diffuse congo red staining within glomerular, vascular and interstitial compartments. Transthyretin and amyloid A staining were negative. Molecular genetic testing identified presence for the c314A>T and c263C>A variant in the LYZ gene. Further testing of an affected parent confirmed the presence of the same LYZ genetic mutations. Lysozyme amyloidosis is a poorly characterised and rarely encountered hereditary form of amyloidosis. We report two cases of lysozyme amyloidosis encountered by our renal service with molecular genetic testing describing two novel genetic mutations. A high index of suspicion must be maintained to ensure appropriate classification of amyloidosis.

Duodenal biopsy: an unexpected confirmatory test for a patient with al-amyloidosis

SSystemic AL- amyloidosis is a disorder of protein folding in which there is extra-cellular accumulation as β pleated fibrillar deposits of monoclonal immunoglobulin light chain fragments. AL- amyloidosis is a rare clinical entity. A presentation with clinical and histological evidence of gastrointestinal amyloidosis is even rare. Here we report primary AL- amyloidosis secondary to lamda (λ) light chain myeloma in a middle-aged woman who presented with nephrotic syndrome and motor peripheral neuropathy.