Truncation of the constant domain drives amyloid formation by immunoglobulin light chains.

AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.

Degradation versus fibrillogenesis, two alternative pathways modulated by seeds and glycosaminoglycans.

The mechanism that converts native human transthyretin into amyloid fibrils in vivo is still a debated and controversial issue. Commonly, non-physiological conditions of pH, temperature, or organic solvents are used in in vitro models of fibrillogenesis of globular proteins. Transthyretin amyloid formation can be achieved under physiological conditions through a mechano-enzymatic mechanism involving specific serine proteases such as trypsin or plasmin. Here, we investigate S52P and L111M transthyretin variants, both causing a severe form of systemic amyloidosis mostly targeting the heart at a relatively young age with heterogeneous phenotype among patients. Our studies on thermodynamics show that both proteins are significantly less stable than other amyloidogenic variants. However, despite a similar thermodynamic stability, L111M variant seems to have enhanced susceptibility to cleavage and a lower tendency to form fibrils than S52P in the presence of specific proteases and biomechanical forces. Heparin strongly enhances the fibrillogenic capacity of L111M transthyretin, but has no effect on the S52P variant. Fibrillar seeds similarly affect the fibrillogenesis of both proteins, with a stronger effect on the L111M variant. According to our model of mechano-enzymatic fibrillogenesis, both full-length and truncated monomers, released after the first cleavage, can enter into fibrillogenesis or degradation pathways. Our findings show that the kinetics of the two processes can be affected by several factors, such as intrinsic amyloidogenicity due to the specific mutations, environmental factors including heparin and fibrillar seeds that significantly accelerate the fibrillogenic pathway.

Human wild-type and D76N ß2-microglobulin variants are significant proteotoxic and metabolic stressors for transgenic C. elegans.

ß2-microglobulin (ß2-m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild-type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N ß2-m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans (C. elegans) strain expressing human WT ß2-m at high concentrations, mimicking the condition that underlies dialysis-related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N ß2-m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with ß2-m levels rather than with the presence of mutations, being more pronounced in WT ß2-m worms. ß2-m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT ß2-m at high concentration compared to D76N ß2-m worms. Altogether, these data show that ß2-m is a proteotoxic protein in vivo also in its wild-type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary ß2-m amyloidosis (high levels of non-mutated ß2-m vs. normal levels of variant ß2-m) and provide important clues on the molecular bases of these human diseases.

The Protein Network in Subcutaneous Fat Biopsies from Patients with AL Amyloidosis: More Than Diagnosis?

AL amyloidosis is caused by the misfolding of immunoglobulin light chains leading to an impaired function of tissues and organs in which they accumulate. Due to the paucity of -omics profiles from undissected samples, few studies have addressed amyloid-related damage system wide. To fill this gap, we evaluated proteome changes in the abdominal subcutaneous adipose tissue of patients affected by the AL isotypes κ and λ. Through our retrospective analysis based on graph theory, we have herein deduced new insights representing a step forward from the pioneering proteomic investigations previously published by our group. ECM/cytoskeleton, oxidative stress and proteostasis were confirmed as leading processes. In this scenario, some proteins, including glutathione peroxidase 1 (GPX1), tubulins and the TRiC complex, were classified as biologically and topologically relevant. These and other results overlap with those already reported for other amyloidoses, supporting the hypothesis that amyloidogenic proteins could induce similar mechanisms independently of the main fibril precursor and of the target tissues/organs. Of course, further studies based on larger patient cohorts and different tissues/organs will be essential, which would be a key point that would allow for a more robust selection of the main molecular players and a more accurate correlation with clinical aspects.

Calcium Binds to Transthyretin with Low Affinity.

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.

Recommendations for addressing the translational gap between experimental and clinical research on amyloid diseases.

This paper is a report of recommendations for addressing translational challenges in amyloid disease research. They were developed during and following an international online workshop organized by the LINXS Institute of Advanced Neutron and X-Ray Science in March 2021. Key suggestions include improving cross-cultural communication between basic science and clinical research, increasing the influence of scientific societies and journals (vis-à-vis funding agencies and pharmaceutical companies), improving the dissemination of negative results, and strengthening the ethos of science.

Amyloid Formation by Globular Proteins: The Need to Narrow the Gap Between in Vitro and in Vivo Mechanisms.

The globular to fibrillar transition of proteins represents a key pathogenic event in the development of amyloid diseases. Although systemic amyloidoses share the common characteristic of amyloid deposition in the extracellular matrix, they are clinically heterogeneous as the affected organs may vary. The observation that precursors of amyloid fibrils derived from circulating globular plasma proteins led to huge efforts in trying to elucidate the structural events determining the protein metamorphosis from their globular to fibrillar state. Whereas the process of metamorphosis has inspired poets and writers from Ovid to Kafka, protein metamorphism is a more recent concept. It is an ideal metaphor in biochemistry for studying the protein folding paradigm and investigating determinants of folding dynamics. Although we have learned how to transform both normal and pathogenic globular proteins into fibrillar polymers in vitro, the events occurring in vivo, are far more complex and yet to be explained. A major gap still exists between in vivo and in vitro models of fibrillogenesis as the biological complexity of the disease in living organisms cannot be reproduced at the same extent in the test tube. Reviewing the major scientific attempts to monitor the amyloidogenic metamorphosis of globular proteins in systems of increasing complexity, from cell culture to human tissues, may help to bridge the gap between the experimental models and the actual pathological events in patients.

AA amyloidosis without systemic inflammation: when clinical evidence validates predictions of experimental medicine.

Amyloid A (AA) amyloidosis is a well-known consequence of chronic inflammatory diseases in which elevated plasma concentrations of serum amyloid A result in amyloid aggregation and organ damage. In this issue, Sikora et al. report, for the first time, an inherited form of AA amyloidosis occurring in the absence of systemic inflammation. This finding may provide novel insights into the pathogenesis of AA amyloidosis, allowing researchers to further dissect the role of inflammation from that of serum amyloid A.

[Arthroscopically assisted minimally invasive symphysioplasty for the treatment of pubic related groin pain]. / Arthroskopisch assistierte minimal-invasive Symphysioplastik zur Behandlung des schambeinbezogenen Leistenschmerzes.

OBJECTIVE: Therapy of pubic related groin pain via minimally invasive symphysioplasty. INDICATIONS: Therapy of refractory pubic related groin pain based on osteitis pubis. CONTRAINDICATIONS: Groin pain from causes other than pubic related groin pain. SURGICAL TECHNIQUE: After a minimally invasive approach, an incision in the anterior capsule is made while protecting the dorsal capsule parts and the arcuate pubic ligament. The symphysis end plates are remodeled arthroscopically assisted using a surgical burr. The newly created pubic symphysis joint is filled with autogenous fibrin to support the formation of a new discus interpubicus. POSTOPERATIVE MANAGEMENT: Partial weight-bearing for 4 weeks with 20 kg using crutches is recommended. During the first 4 weeks the range of motion should be restricted. RESULTS: Since 2010, 10 athletes (7 men, 3 women; average age 34.1 ± 7.8 (23-47) years) have undergone arthroscopically assisted minimally invasive symphysioplasty and treatment of femoroacetabular impingement syndrome. The average follow-up time was 5.1 (2-9) years. All patients returned to their sport level. The mean preoperative Nonarthritic Hip Score (NAHS) of 64.4 ± 15.1 (32.1-86.5) points improved to a mean postoperative NAHS of 91.4 ± 9.8 (62.4-98.75) points (p < 0.0001). The average patient satisfaction (scale 0 to 10; 10 highest satisfaction) was 9.8 ± 0.4 (9-10).

The corona of protein-gold nanoparticle systems: the role of ionic strength.

The nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with ß2-microglobulin (ß2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a ß2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between ß2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard" interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between ß2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength.