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The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Å resolution by molecular replacement. Crystal belongs to space group I2 1 2 1 2 1 (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo . The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro . In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro , is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.