How antibodies fold

128

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.ER proteostasis | amyloid L ight-chain amyloidosis (AL) afflicts 8-10 people per million per year, making it the most prominent systemic amyloid disease (1). AL is a gain-of-toxic function disease driven by a clonally expanded plasma cell that secretes amyloidogenic Ig light chains (LCs). These amyloidogenic LCs undergo extracellular misfolding and aggregation into proteotoxic soluble oligomers and amyloid fibrils that interact with distal tissues such as the kidney, heart, and gastrointestinal tract, leading to organ dysfunction and ultimately death by unknown proteotoxicity mechanism(s) (2).The majority of AL patients must combat both a cancer (i.e., the clonally expanded plasma cell) and LC aggregation-associated proteotoxicity. The standard treatment for AL patients is chemotherapy (often combined with stem cell transplant) to eliminate the cancerous plasma cell population (3, 4). The proteasome inhibitor bortezomib, which takes advantage of the stress sensitivity of aggressively proliferating plasma cells, has transformed chemotherapy effectiveness (5-7). Regardless, ∼30% of AL patients with substantial cardiac or renal LC proteotoxicity are too ill at diagnosis to tolerate chemotherapeutics (8-10). Thus, new strategies to reduce LC organ proteotoxicity must be developed to allow more AL patients to take advantage of chemotherapy.LC aggregation requires conformational changes and a sufficient concentration of misfolded LC in plasma, which determine the rate and extent of its concentration-dependent aggregation. Over 500 distinct LC s...

Section: Resultsmentioning

confidence: 72%

Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

Cooley¹,

Ryno²,

Plate³

et al. 2014

128

“…1 B and C). In many mammalian antibody domains, this proline is in the cis state in the native structure, and thus, because of its slow isomerization, it leads to the greater population of folding intermediates, decisively influencing the domain folding reaction (22,23). In the C1 and C3 domain, a cisproline residue is located in this conserved position; however, no proline is found at the corresponding position in C2 or C4 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, we found the small helix between strands e and f of evolutionary more recent antibody domains to also be present in C1-C4. A highly conserved tryptophan in this helix interacts with an almost equally conserved aromatic residue located C terminally, arguing that this motif is evolutionary ancient and contributes to the stability and robust folding pathway of antibody domains, as opposed to some other Ig domains, which usually do not possess these elements (23,39,40).…”

Section: Discussionmentioning

confidence: 99%

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Feige

Gräwert

Marcinowski

et al. 2014

Self Cite

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules. protein evolution | antibody structure | protein folding | protein engineering

“…Whereas the structure and assembly of IgG is well-studied (19), for IgM we largely lack detailed structural information. Here we present a hybrid approach using X-ray crystallography, NMR, and SAXS to solve the structures of the individual domains of the mouse IgM Fc (Cμ2, Cμ3, and Cμ4) and to reconstruct the Fc oligomer.…”

Section: Discussionmentioning

confidence: 99%

High-resolution structures of the IgM Fc domains reveal principles of its hexamer formation

Müller

Gräwert

Kern

et al. 2013

Self Cite

IgM is the first antibody produced during the humoral immune response. Despite its fundamental role in the immune system, IgM is structurally only poorly described. In this work we used X-ray crystallography and NMR spectroscopy to determine the atomic structures of the constant IgM Fc domains (Cµ2, Cµ3, and Cµ4) and to address their roles in IgM oligomerization. Although the isolated domains share the typical Ig fold, they differ substantially in dimerization properties and quaternary contacts. Unexpectedly, the Cµ4 domain and its C-terminal tail piece are responsible and sufficient for the specific polymerization of Cµ4 dimers into covalently linked hexamers of dimers. Based on small angle X-ray scattering data, we present a model of the ring-shaped Cµ4 structure, which reveals the principles of IgM oligomerization.