Development of Conformational Antibodies to Detect Bcl-xL’s Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells

Abstract

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

Chiral Linked Systems as a Model for Understanding D-Amino Acids Influence on the Structure and Properties of Amyloid Peptides

“…As mentioned in the introduction, the nature of neurodegenerative diseases, including Alzheimer’s disease, is largely related to amyloid Aβ42 [ 1 , 2 , 3 , 4 ]. At the same time, research of this amyloid is carried out in several directions, on the one hand, the toxicity of oligomers of this amyloid relative to brain cells is studied [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ], and on the other hand, its structure is explored [ 3 , 4 , 9 ]. One of the main questions: how does the structure of this peptide change when of L-amino acids are replaced by D-analog?…”

“…The choice of NSAIDs is not accidental, since the derivatives of these drugs are now being tested as inhibitor of γ-secretase-a complex of enzymes involved in the synthesis of Aβ42 from precursor of amyloid protein (APP) [18,19]. The mechanisms of the Aβ42 monomer action and its aggregated forms have been studied in cell models, while the primary cause of the formation of aggregates and their relationship with the inclusion of D-amino acids in biomolecules remains to be studied [3,4,[9][10][11]. This review describes first results on the influence of D-amino acids on the structure and properties, obtained in the study of model systems (Figure 1).…”

“…The mechanisms of the Aβ42 monomer action and its aggregated forms have been studied in cell models, while the primary cause of the formation of aggregates and their relationship with the inclusion of D-amino acids in biomolecules remains to be studied [ 3 , 4 , 9 , 10 , 11 ]. This review describes first results on the influence of D-amino acids on the structure and properties, obtained in the study of model systems ( Figure 1 ).…”

Understanding Amyloid Structures and Disease: A Continuing Challenge in Health Research

“…In addition, Bcl-xL, a pro-survival protein involved in apoptosis regulation [ 11 ], has been previously reported to form various types of fibers, from native to amyloid conformations. By developing specific monoclonal antibodies (mAbs) directed against amyloidogenic conformations of Bcl-xL, Gonneaud et al [ 12 ] were able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. Therefore, these mAbs may further help in developing new diagnostics and therapies, by exploiting Bcl-xL as a strategic target for NDDs.…”

Inducible fold‐switching as a mechanism to fibrillate pro‐apoptotic BCL‐2 proteins

“…It is important to note that β-sheet fibrillation of the BCL-2 protein B-cell lymphomaextra-large (BCL-x L ) alone was reported, and some antibodies were developed that could detect BCL-x L fibrils in vivo. [27,28] In contrast to HN and BAX or BID fibrils, these BCL-x L fibrils can take days to form at 37 C in vitro. Unlike BAX and BID which are pro-apoptotic members of the BCL-2 family, BCL-x L is anti-apoptotic and we showed addition of HN did not cause it to form fibrils.…”