Systemic Amyloid Disease Research Articles

Structural Basis for Monoclonal Antibody Therapy for Transthyretin Amyloidosis.

The disease of transthyretin (TTR) amyloidosis (ATTR) has been known since the 1960s, and during the past 60 or so years, there has been a sustained period of steady discoveries that have led to the current model of ATTR pathogenesis. More recent research has achieved major advances in both diagnostics and therapeutics for ATTR, which are having a significant impact on ATTR patients today. Aiding these recent achievements has been the remarkable ability of cryo-electron microscopy (EM) to determine high-resolution structures of amyloid fibrils obtained from individual patients. Here, we will examine the cryo-EM structures of transthyretin amyloid fibrils to explore the structural basis of the two monoclonal antibody therapies for ATTR that are in clinical trials, ALXN-2220 and Coramitug, as well as to point out potential applications of this approach to other systemic amyloid diseases.

Understanding and Overcoming Biochemical Diversity in AL Amyloidosis

AbstractAmyloid fibril deposition causes progressive tissue damage and organ failure in the systemic amyloid diseases, and therapies that suppress aggregation lead to clinical benefit. Small molecules that prevent aggregation by binding to precursor proteins are effective for amyloid transthyretin (ATTR) amyloidosis. However, in amyloid light chain (AL) amyloidosis, fibrils are formed by antibody light chains and every patient has a unique protein sequence that aggregates. The highly diverse sequences of these light chains appear to determine whether an individual is at risk of amyloidosis, the distribution of amyloid deposits and the progression of disease. Light chains are therefore challenging drug targets. This review explores the parallels between AL amyloidosis and ATTR amyloidosis to describe the discovery of small molecules that can stabilize light chains. These molecules have potential as therapies for AL amyloidosis, highlighting potential opportunities for drug discovery in other diseases of protein misfolding.

Systemic Amyloid Response in Patients with AL Amyloidosis Assessed By Serial Iodine ( 124I) Evuzamitide PET/CT Imaging

Background: Immunoglobulin light chain (AL) amyloidosis is a systemic disorder. Fibrillar aggregates of monoclonal free light chains, or fragments thereof, can deposit in any organ or tissue. Invariably, more than one anatomic site is involved, including the heart, liver, spleen, kidneys, pancreas, nerves, gastrointestinal tract and vasculature. Cardiac and renal amyloidosis are clinically most ominous; however, amyloid in other sites significantly impacts quality of life and may cause other heretofore unappreciated clinical sequelae. This is also true of the other 17 types of systemic amyloidosis. The degree of amyloid distribution and the tissue-specific amyloid load can only be achieved by nuclear imaging. Moreover, monitoring changes in amyloid load in response to therapy would benefit from whole body quantitative molecular imaging techniques. Here we report a pilot study of eight ( n=8) patients with AL amyloidosis who underwent serial PET/CT imaging using a novel amyloid-binding, iodine-124-labelled peptide, designated iodine-124-evuzamitide. Data from a cohort of ten ( n=10) patients with transthyretin associated (ATTR) amyloidosis are also described for comparison (NCT05968846). Methods: The study plans to enroll 20 ( n=20) patients with AL ( n=10) and ATTR ( n=10) amyloidosis who were previously imaged more than 12 months prior and who had positive uptake of 124I-evuzamitide in the heart. To date, eight ( n=8) patients with AL and ten ( n=10) with ATTR amyloidosis have undergone repeat imaging since 2019. Between imaging sessions, patients with AL received immunotherapy ( n=2/8) or were untreated ( n=6/8). Patients were administered 37 or 74 MBq 124I-evuzamitide, and PET/CT imaging was performed at ~5 h post injection. Clinical chemistry parameters were collected contemporaneously. Images were interpreted by a single reviewer, experienced in evaluating this tracer, for positive uptake. A fully manual 2D region of interest (ROI) analysis was performed on three representative image views of the heart in the transaxial plane as well as the liver, spleen, and kidneys. The blood pool was used as a reference tissue to determine standard uptake value ratios (SUVR mean). Cardiac uptake of radiotracer was compared with serum NT-proBNP measurements. Pearson and Spearman correlations were used to test for associations. Results: The mean time between imaging sessions for patients with AL amyloidosis was 3.21±0.95 years and 2.96±0.87 years for the entire cohort. In the AL amyloid cohort, 88% ( n=7/8) had a reduction in cardiac radiotracer uptake. In one ( n=1) patient, a 92.9% increase in cardiac uptake was seen, which was readily visible in scaled maximum intensity PET images. In this patient, the liver, spleen, and kidney uptake also increased. In contrast to the heart, renal uptake of the radiotracer was increased in 62.5% ( n=5/8) of patients with AL, with three ( n=3) patients showing a >20% increase in the SUVR mean. A strongly positive, significant correlation between the change in the liver and spleen uptake was observed (Pearson r=0.88, p=0.004). The change in the cardiac radiotracer uptake correlated strongly with that of the spleen (Pearson r=0.74, p=0.04). For the entire cohort, AL and ATTR patients ( n=18), there was a significant moderate correlation between the change in cardiac SUVR mean and change in single point serum NTproBNP concentration (Spearman rho=0.57, p=0.01). Conclusion: AL amyloidosis, and indeed all types of systemic amyloid disease, is a complex systemic disorder. The amyloid response to treatment, or disease progression, remains enigmatic as compared to the hematologic and biomarker responses. Appreciating the changes in amyloid load may not only improve clinical management but may also illuminate aspects of pathogenesis. PET/CT imaging using 124I-evuzamitide may, therefore, play a valuable role in understanding, diagnosing, and monitoring changes in organ-specific amyloid load and amyloid response. Additional longitudinal imaging studies are needed to investigate these possibilities further.

Pre-Clinical Characterization of a Novel Fusion Protein (AT-03), with Pan-Amyloid Binding and Removal

Background: Amyloidosis is characterized by extracellular deposition of protein fibrils that can involve any organ or tissue in the body. It is a severe, progressive and often lethal disorder. There are approximately seventeen different systemic amyloid diseases, each associated with the deposition of a specific precursor protein. Toxic amyloid deposits result in significant organ dysfunction and increased morbidity and mortality. Reduction and removal of tissue amyloid deposits could restore organ function, improve quality of life and increase life expectancy for patients. We have developed and characterized a fusion protein consisting of serum amyloid protein (SAP) linked to a single chain human IgG1 Fc domain, designated AT-03. SAP is a pentameric serum protein that is ubiquitously associated with amyloid deposits. The high affinity and specificity for amyloid resulted in the use of radio-iodinated SAP as an imaging agent for the non-invasive detection of systemic amyloid. The SAP domain of the fusion protein targets the amyloid deposits delivering Fc domains to stimulate amyloid removal via macrophage mediated phagocytosis.Methods: Binding of AT-03 to ATTR and AL fibrils and extracts was assessed by ELISA. Ex vivo phagocytosis of amyloid substrates was studied using activated human THP-1 cells. The biodistribution of AT-03 in murine models of AA, AApoA2 and AL amyloidosis was evaluated immunohistochemically or by SPECT imaging and microautoradiography. Clearance of AA amyloidosis in response to AT-03 therapy was accessed in a mouse model using histological scoring of amyloid burden.Results: AT-03 bound amyloid AL and ATTR substrates with sub nanomolar EC50 values. Systemic administration of AT-03 resulted in specific binding to diverse forms of amyloid in the heart, kidney, liver and/or spleen in multiple animal models. Opsonization of amyloid deposits with AT-03 in vitro resulted in a dose dependent phagocytosis by activated THP-1 cells. A single IV dose of AT-03 resulted in significant reduction of splenic AA amyloid within 14 days post-injection.Conclusions: We have developed a novel fusion protein with sub nanomolar pan amyloid reactivity that specifically binds diverse forms of amyloid in multiple organs and tissues in vivo and can effectuate amyloid removal. The data indicates that AT-03 may serve as a novel therapeutic for the removal of systemic amyloid deposits of diverse types. AT-03 is currently being developed for clinical evaluation. DisclosuresSirac: Attralus, Inc: Patents & Royalties, Research Funding. Jaccard: Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Abbvie: Honoraria. Bridoux: AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy. Bell: Attralus Inc: Current Employment. Guthrie: Attralus Inc: Current Employment. Selvarajah: Attralus Inc: Current Employment. Kennel: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder. Wall: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder, Research Funding.

Ocular Involvement in Hereditary Amyloidosis.

The term amyloidosis describes a group of rare diseases caused by protein conformation abnormalities resulting in extracellular deposition and accumulation of insoluble fibrillar aggregates. So far, 36 amyloid precursor proteins have been identified, and each one is responsible for a specific disease entity. Transthyretin amyloidosis (ATTRv) is one of the most common forms of systemic and ocular amyloidosis, due to the deposition of transthyretin (TTR), which is a transport protein mainly synthesized in the liver but also in the retinal pigment epithelial cells. ATTRv amyloidosis may be misdiagnosed with several other conditions, resulting in a significant diagnostic delay. Gelsolin and keratoepithelin are other proteins that, when mutated, are responsible for a systemic amyloid disease with significant ocular manifestations that not infrequently appear before systemic involvement. The main signs of ocular amyloid deposition are in the cornea, irido-corneal angle and vitreous, causing complications related to vasculopathy and neuropathy at the local level. This review aims at describing the main biochemical, histopathological and clinical features of systemic amyloidosis associated with eye involvement, with particular emphasis on the inherited forms. We discuss currently available treatments, focusing on ocular involvement and specific ophthalmologic management and highlighting the importance of a prompt treatment for the potential sight-threatening complications derived from amyloid deposition in ocular tissues.

Characterisation of the structural, dynamic and aggregation properties of the W64R amyloidogenic variant of human lysozyme

The accumulation in vital organs of amyloid fibrils made of mutational variants of lysozyme (HuL) is associated with a human systemic amyloid disease. The detailed comparison of the in vitro properties of the I56T and D67H amyloidogenic variants to those of the T70N non-amyloidogenic variant and the wild-type (WT) protein suggested that the deposition of large amounts of aggregated disease-related lysozyme variants is initiated by the formation of transient intermediate species. The ability to populate such intermediates is essentially due to the destabilisation of the protein and the loss of the global structural cooperativity under physiologically relevant conditions. Here, we report the characterisation of a third naturally occurring amyloidogenic lysozyme variant, W64R, in comparison with the I56T and WT proteins. The X-ray crystal structure of the W64R variant at 1.15 Å resolution is very similar to that of the WT protein; a few interactions within the β-domain and at the interface between the α- and β-domains differ, however, from those in the WT protein. Consequently, the W64R mutation destabilizes the protein to an extent that is similar to that observed for the I56T and D67H mutations. The ΔG°NU(H2O) is reduced by 24 kJ·mol−1 and the Tm is about 12 °C lower than that of the WT protein. Under native conditions, the W64R and I56T proteins are readily digested by proteinase K, while the WT protein remains intact. These results suggest that the two variant proteins transiently populate similar partially unfolded states in which proteinase K cleavage sites are accessible to the protease. Moreover, the in vitro aggregation properties of the W64R protein are similar to those of the I56T variant. Altogether, these results indicate that the properties of the W64R protein are astonishingly similar to those of the I56T variant. They further corroborate the idea that HuL variants associated with the disease are those whose stability and global structural cooperativity are sufficiently reduced to allow the formation of aggregation prone partially folded intermediates under physiological conditions.

Does protein aggregation drive postmitotic tissue degeneration?

Pharmacological evidence, from clinical trials where patients with systemic amyloid diseases are treated with disease-modifying therapies, supports the notion that protein aggregation drives tissue degeneration in these disorders. The protein aggregate structures driving tissue pathology and the commonalities in etiology between these diseases and Alzheimer's disease are under investigation.

Starting at the beginning: endoplasmic reticulum proteostasis and systemic amyloid disease.

Systemic amyloid diseases are characterized by the deposition of an amyloidogenic protein as toxic oligomers and amyloid fibrils on tissues distal from the site of protein synthesis. Traditionally, these diseases have been viewed as disorders of peripheral target tissues where aggregates are deposited, and toxicity is observed. However, recent evidence highlights an important role for endoplasmic reticulum (ER) proteostasis pathways within tissues synthesizing and secreting amyloidogenic proteins, such as the liver, in the pathogenesis of these disorders. Here, we describe the pathologic implications of ER proteostasis and its regulation on the toxic extracellular aggregation of amyloidogenic proteins implicated in systemic amyloid disease pathogenesis. Furthermore, we discuss the therapeutic potential for targeting ER proteostasis to reduce the secretion and toxic aggregation of amyloidogenic proteins to mitigate peripheral amyloid-associated toxicity involved in the onset and progression of systemic amyloid diseases.

Preliminary Phase 1 Data on the Safety and Efficacy of a Novel PET Radiotracer, 124I-p5+14, for Imaging Systemic Amyloidosis

Background: Systemic immunoglobulin light chain-associated amyloidosis (AL) is a rare disorder associated with plasma cell dyscrasias and the production of monoclonal free light chain proteins. Pathology results from the extracellular deposition of proteinaceous (amyloid) fibrils in association with proteoglycans and other serum derived proteins. The amyloid accumulates in abdominothoracic organs, notably the heart, liver, spleen, kidneys, as well as peripheral nerves leading to organ dysfunction and significant morbidity. At present, there are no radiotracers approved in the US for the non-invasive quantitative measurement of systemic AL amyloid disease. We have developed a synthetic polybasic peptide radiotracer, designated 124I-p5+14, which has been shown, in preclinical assays, to bind many forms of amyloid, including AL, ATTR, and ALECT2 (Wall, J.S. et al. (2015) Molecules, 20, 7657). The peptide binds, through multivalent electrostatic interactions, to the hypersulfated heparan sulfate glycosaminoglycans and potentially to the fibrillar constituents of amyloid deposits. Using SPECT and PET imaging of murine systemic amyloidosis, we have demonstrated the specific interaction of radioiodinated peptide p5+14 with amyloid in abdominothoracic organs and tissues. No detectable binding of p5+14 with amyloid-free tissues was observed. In light of these data, p5+14 was prepared for a first-in-human PET imaging study using iodine-124-labeled peptide (NCT 03678259). Methods: Patients >18 years of age with biopsy proven amyloidosis with adequate renal function and not requiring heparin therapy are eligible. Subjects receive <2 mg of 124I-p5+14 (<2 mCi) administered as a single IV bolus. PET/CT images for the initial cohort (n = 3) were acquired at 25 min, 50 min, 2 h, 3 h, 6h, 24 h and 48 h post injection. The second cohort of patients are imaged at ~6 h and 24 h post injection. Image data are acquired using a Biograph 16 PET/CT scanner using a low dose CT and 5 min bed positions for PET imaging to cover the full CT field of view. The primary endpoint includes safety and dosimetry estimation with a secondary endpoint of efficacy (biodistribution and organ-specific retention) of 124I-p5+14. Patients receive follow up contact by study staff on days 9 and 28 for safety and symptom assessment. Results: To date, 10 patients have been dosed and evaluated. No serious adverse events of any grade were noted. Organ-specific and whole body effective dosimetry were calculated using time activity curves generated from the initial cohort. The gender-averaged mean whole body effective dose attributed to 124I-p5+14 injection was estimated to be ~0.5 mSv/MBq. Blood pool clearance of the 124I-p5+14 was analyzed using a two phase exponential equation which yielded an excellent fit to the data (R2 > 0.99 for all data), with estimated fast half-life values of 11.8 min - 22.4 min, and a corresponding elimination half-life of between 667.8 min and 746.7 min. Analysis of PET images indicated retention of 124I-p5+14 in the heart, kidneys, liver, spleen, pancreas, bone marrow, lung, and adrenal gland of AL patients (Fig. 1 (A) Cardio-renal AL, and (B) Hepatosplenic AL amyloidosis). Cardiac uptake of the radiotracer was observed in 80% of AL patients (n = 5) who received ≥ 1 mCi 124I-p5+14, with a mean myocardium:blood pool ratio of 2.3 ± 0.5 in positive images. Additionally, liver, spleen and kidney retention of 124I-p5+14 was observed in 20%, 40%, and 60% of AL patients, respectively. Furthermore, retention of the radiotracer has been indicated in the heart, spleen and kidney in patients with ATTR and ALECT2--associated amyloidoses. Conclusion: Initial PET/CT image data indicate that 124I-p5+14 can provide quantitative detection of systemic AL amyloidosis in multiple organ systems and may have general utility in detecting and monitoring amyloid burden in many forms of amyloidosis. Acknowledgments: This study was supported in part by the National Heart Lung and Blood Institute, National Institutes of Health, through the Science Moving TowArds Research Translation and Therapy (SMARTT) program via the following contracts: HHSN268201600011C, HHSN268201600012C, and HHSN268201600014C, as well as by contributions from Gerdau to the ACTP Gift Fund at the UTGSM and support from UHS. In addition, we thank Michael Stabin, PhD, Brett Hines, Carmella Moody, PhD and Derry Ridgway, MD and the many patients for their support. Figure 1 Disclosures Wall: Solex LLC: Equity Ownership; University of Tennessee Research Foundation: Patents & Royalties. Stuckey:Solex LLC: Equity Ownership. Martin:Solex LLC: Equity Ownership. Richey:Solex LLC: Equity Ownership. Ramchandren:Sandoz-Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Pharmacyclics LLC, an Abbvie company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Kennel:University of Tennessee Research Foundation: Patents & Royalties; Solex LLC: Equity Ownership.

Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein

Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. We employ a quantitative interactomics platform to define how ATF6 activation reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with light-chain amyloidosis (AL). Using this platform, we show that ATF6 activation increases the targeting of this destabilized LC to a subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion. Our results define a molecular basis for the ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.

Incomplete Refolding of Antibody Light Chains to Non-Native, Protease-Sensitive Conformations Leads to Aggregation: A Mechanism of Amyloidogenesis in Patients?

Genetic, biochemical, and pharmacologic evidence supports the hypothesis that conformationally altered or misfolded protein states enable aggregation and cytotoxicity in the systemic amyloid diseases. Reversible structural fluctuations of natively folded proteins are involved in the aggregation of many degenerative disease associated proteins. Herein, we use antibody light chains (LCs) that form amyloid fibrils in AL amyloidosis to consider an alternative hypothesis of amyloidogenesis: that transient unfolding and incomplete extracellular refolding of secreted proteins can lead to metastable, alternatively folded states that are more susceptible to aggregation or to endoproteolysis that can release aggregation-prone fragments. Refolding of full-length λ6a LC dimers comprising an interchain disulfide bond from heat- or chaotrope-denatured ensembles in buffers yields the native dimeric state as well as alternatively folded dimers and aggregates. LC variants lacking an interchain disulfide bond appear to refold fully reversibly to the native state. The conformation of a backbone peptidyl-proline amide in the LC constant domain, which is cis in the native state, may determine whether the LC refolds back to the native state. A proline to alanine (P147A) LC variant, which cannot form the native cis-amide conformation, forms amyloid fibrils from the alternatively folded ensemble, whereas all the full-length λ6a LCs we have studied to date do not form amyloid under analogous conditions. P147A LC variants are susceptible to endoproteolysis by thrombin, enabling amyloidogenesis of the fragments released. Thus, non-native LC structural ensembles containing a tyrosine 146-proline 147 trans-amide bond can initiate and propagate amyloid formation, either directly or after aberrant endoproteolysis.

Variation of amino acid sequences of serum amyloid a (SAA) and immunohistochemical analysis of amyloid a (AA) in Japanese domestic cats.

Amyloid A (AA) amyloidosis, a fatal systemic amyloid disease, occurs secondary to chronic inflammatory conditions in humans. Although persistently elevated serum amyloid A (SAA) levels are required for its pathogenesis, not all individuals with chronic inflammation necessarily develop AA amyloidosis. Furthermore, many diseases in cats are associated with the elevated production of SAA, whereas only a small number actually develop AA amyloidosis. We hypothesized that a genetic mutation in the SAA gene may strongly contribute to the pathogenesis of feline AA amyloidosis. In the present study, genomic DNA from four Japanese domestic cats (JDCs) with AA amyloidosis and from five without amyloidosis was analyzed using polymerase chain reaction (PCR) amplification and direct sequencing. We identified the novel variation combination of 45R-51A in the deduced amino acid sequences of four JDCs with amyloidosis and five without. However, there was no relationship between amino acid variations and the distribution of AA amyloid deposits, indicating that differences in SAA sequences do not contribute to the pathogenesis of AA amyloidosis. Immunohistochemical analysis using antisera against the three different parts of the feline SAA protein—i.e., the N-terminal, central, and C-terminal regions—revealed that feline AA contained the C-terminus, unlike human AA. These results indicate that the cleavage and degradation of the C-terminus are not essential for amyloid fibril formation in JDCs.

Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to β-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

A case of localized amyloidosis of the eyelid misdiagnosed as recurrent chalazion

Localized amyloidosis of the eyelid is uncommon and is classically associated with systemic manifestations. We present an interesting case of a localized eyelid mass misdiagnosed as a recurrent chalazion presenting in an 85-year-old Saudi gentleman with no definite associate findings suggestive of an underlying systemic amyloid disease. Debulking surgery was subsequently performed. Proper diagnosis was reached based on the histopathologic examination of the excised tissue, which demonstrated the typical Congo red staining of the amyloid deposits.

Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells

Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.

Expanding the spectrum of systemic amyloid diseases: a new hint from the kidney

Proteomics-based assays have substantially improved diagnostic accuracy in systemic amyloidosis and have led to the discovery of novel amyloidogenic proteins. The observation that human apolipoproteins are increasingly represented is intriguing and highlights the complex, and partially known, mutual effects of the misfolded proteins and lipids. Dasari etal. report on the first series of patients affected by the recently described apolipoprotein A-IV amyloidosis, detailing its peculiar clinical and pathologic findings and diagnostic pitfalls.

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

Endoplasmic reticulum quality control and systemic amyloid disease: Impacting protein stability from the inside out.

The endoplasmic reticulum (ER) is responsible for regulating proteome integrity throughout the secretory pathway. The ER protects downstream secretory environments such as the extracellular space by partitioning proteins between ER protein folding, trafficking, and degradation pathways in a process called ER quality control. In this process, ER quality control factors identify misfolded, aggregation-prone protein conformations and direct them toward ER protein folding or degradation, reducing their secretion to the extracellular space where they could further misfold or aggregate into proteotoxic conformations. Despite the general efficiency of ER quality control, many human diseases, such as the systemic amyloidoses, involve aggregation of destabilized, aggregation-prone proteins in the extracellular space. A common feature for all systemic amyloid diseases is the ability for amyloidogenic proteins to evade ER quality control and be efficiently secreted. The efficient secretion of these amyloidogenic proteins increases their serum concentrations available for the distal proteotoxic aggregation characteristic of these diseases. This indicates that ER quality control, and the regulation thereof, is a critical determinant in defining the onset and pathology of systemic amyloid diseases. Here, we discuss the pathologic and potential therapeutic relationship between ER quality control, protein secretion, and distal deposition of amyloidogenic proteins involved in systemic amyloid diseases. Furthermore, we present evidence that the unfolded protein response, the stress-responsive signaling pathway that regulates ER quality control, is involved in the pathogenesis of systemic amyloid diseases and represents a promising emerging therapeutic target to intervene in this class of human disease.

Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis.

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

Probing the influence of citrate-capped gold nanoparticles on an amyloidogenic protein.

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, β2-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of β2-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process.