The potential of lumbrokinase and serratiopeptidase for the degradation of Aß 1-42 peptide - an in vitro and in silico approach.

BACKGROUND: Alzheimer's disease (AD) is diagnosed with the deposition of insoluble ß-amyloid (Aß) peptides in the neuropil of the brain leading to dementia. The extracellular deposition of the fibrillar Aß peptide on the neurons is known as senile plaques. Therefore, Aß degradation and clearance from the human body is a promising therapeutic approach in the medication of AD. METHODS: In the current study, the enzyme lumbrokinase (LK) was extracted and purified from earthworm and its activity was utilized toward Aß 1-42 amyloids degradation in vitro alongside with an additional enzyme serratiopeptidase (SP) considering nattokinase (NK) as a standard. RESULTS: The output of this study revealed that preformed Aß 1-42 amyloids was disintegrated by both LK and SP, as demonstrated from fluorescence assay using Thioflavin T dye. In addition, dynamic light scattering study revealed the lower size of the preformed fibrils Aß 1-42 at various time intervals after incubation with the enzymes LK and SP. Furthermore, in silico approach showed high affinity thermodynamically favorable interaction of LK as well as SP toward Aß 1-42 amyloid. Finally, the toxicity of degraded preformed Aß 1-42 amyloid was assessed by MTT assay which showed reduced toxicity of enzyme treated Aß 1-42 amyloid compared to only Aß 1-42 amyloid. CONCLUSION: The findings of the present study indicated that LK and SP, not only had Aß 1-42 amyloid degrading potential, but also could reduce the toxicity which can make them a suitable drug candidate for AD. Furthermore, the in vivo studies are needed to be executed in future.

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.