Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine.

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.

Exploring the therapeutic potential of Rab11: A comprehensive study on its effectiveness in alleviating rotenone-induced molecular pathogenesis of Parkinson's disease in SH-SY5Y cells and its synergistic application with L-DOPA in Drosophila models.

Dysfunctional mitophagy contributes to Parkinson's disease (PD) by affecting dopamine-producing neurons. Mutations in parkin and pink1 genes, linked to familial PD, impede the removal of damaged mitochondria. Previous studies suggested Rab11's involvement in mitophagy alongside Parkin and Pink1. Additionally, mitochondria-endoplasmic reticulum contact sites (MERCS) regulate cellular functions, including mitochondrial quality control and calcium regulation. Our study explored whether activating mitophagy triggers the unfolded protein response and ER stress pathway in SH-SY5Y human cells. We induced a PD-like state by exposing undifferentiated SH-SY5Y cells to rotenone, an established PD-inducing agent. This led to reduced Rab11 and PERK- expression while increasing ATP5a, a mitochondrial marker, when Rab11 was overexpressed. Our findings suggest that enhancing endosomal trafficking can mitigate ER stress by regulating mitochondria, rescuing cells from apoptosis. Furthermore, we assessed the therapeutic potential of Rab11, both alone and in combination with L-Dopa, in a Drosophila PD model. In summary, our research underscores the role of mitophagy dysfunction in PD pathogenesis, highlighting Rab11's importance in alleviating ER stress and preserving mitochondrial function. It also provides insights into potential PD management strategies, including the synergistic use of Rab11 and L-Dopa.

Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut.

Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N- and C-terminal molecular halves with the ability to bind two Fe(3+) ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apo-lactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.