A constant domain mutation in a patient-derived antibody light chain reveals principles of AL amyloidosis.

Light chain (AL) amyloidosis is a debilitating disease in which mutant antibody light chains (LC), secreted by aberrant plasma cell clones, misfold and form insoluble fibrils, which can be deposited in various organs. In the majority of cases, the fibrillar deposits consist of LC variable domains (VL) containing destabilizing mutations compared to their germline counterparts. This is also true for the patient LC FOR005. However, this pathogenic LC sequence contains an additional mutation in the constant domain (CL). The mechanistic impact of CL mutations is not yet understood in the context of AL amyloidosis. Our analysis reveals that the FOR005 CL mutation influences the amyloid pathway in specific ways: (1) folding and stability of the patient CL domain are strongly impaired; (2) the mutation disrupts the LC dimer interface and weakens dimerization; (3) the CL mutation promotes proteolytic cleavage of the LC monomers resulting in an isolated, amyloidogenic VL domain while dimeric LCs are not cleaved. The enhanced proteolysis rates and the inability of full-length LCs to form amyloid fibrils even in the presence of a destabilized CL domain support a model for AL amyloidosis in which the CL domain plays a protective role and in which proteolytic cleavage precedes amyloid formation.

Antibodies gone bad - the molecular mechanism of light chain amyloidosis.

Light chain amyloidosis (AL) is a systemic disease in which abnormally proliferating plasma cells secrete large amounts of mutated antibody light chains (LCs) that eventually form fibrils. The fibrils are deposited in various organs, most often in the heart and kidney, and impair their function. The prognosis for patients diagnosed with AL is generally poor. The disease is set apart from other amyloidoses by the huge number of patient-specific mutations in the disease-causing and fibril-forming protein. The molecular mechanisms that drive the aggregation of mutated LCs into fibrils have been enigmatic, which hindered the development of efficient diagnostics and therapies. In this review, we summarize our current knowledge on AL amyloidosis and discuss open issues.

Tailored Pyridoxal Probes Unravel Novel Cofactor-Dependent Targets and Antibiotic Hits in Critical Bacterial Pathogens.

Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate-dependent enzymes (PLP-DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP-DEs in clinically relevant pathogens including challenging Gram-negative strains. Putative PLP-DEs with unknown function were exemplarily characterized via in-depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP-dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP-DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

Dissection of the amyloid formation pathway in AL amyloidosis.

In antibody light chain (AL) amyloidosis, overproduced light chain (LC) fragments accumulate as fibrils in organs and tissues of patients. In vitro, AL fibril formation is a slow process, characterized by a pronounced lag phase. The events occurring during this lag phase are largely unknown. We have dissected the lag phase of a patient-derived LC truncation and identified structural transitions that precede fibril formation. The process starts with partial unfolding of the VL domain and the formation of small amounts of dimers. This is a prerequisite for the formation of an ensemble of oligomers, which are the precursors of fibrils. During oligomerization, the hydrophobic core of the LC domain rearranges which leads to changes in solvent accessibility and rigidity. Structural transitions from an anti-parallel to a parallel ß-sheet secondary structure occur in the oligomers prior to amyloid formation. Together, our results reveal a rate-limiting multi-step mechanism of structural transitions prior to fibril formation in AL amyloidosis, which offers, in the long run, opportunities for therapeutic intervention.

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.