Ramirez-Alvarado, Marina

Key Points Mass spectrometry is a high-throughput, low-resource technique that can identify immunoglobulin variable region gene from tissue specimens. IGVL gene usage is restricted and different between systemic and localized AL and only partially explains organ tropism in this disease.

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 [ NLGM 1]) 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 NLGM 1 (1:10 ratio for LC : NLGM 1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM 1, or NLGM 1 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 NLGM 1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM 1. NLGM 1 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 NLGM 1. 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.

Abstract BACKGROUND Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions. METHODS Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment. RESULTS Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%). CONCLUSIONS The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow–based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration.

AbstractLight 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.

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.

Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR) do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis.

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.