Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.05.15.540776

Cellular Bioenergetics Effects of Sirolimus on the Gonads of BALB/c Mice

Narchi, H.·Al Bawardi, A.·Thachillath, P.·Souid, A.-K.
View original source ↗Export metadata ↓

Abstract

BackgroundSirolimus, an immunosuppressive drug widely used in organ transplantation, inhibits the mechanistic target of rapamycin (mTOR), decreases cellular respiration, stops cell cycle progression and causes apoptosis. It has been linked to cytotoxicity in the gonads, but the cause is unknown. AimsTo understand the effects of sirolimus on the gonads by measuring cellular respiration and ATP as indicators of cellular damage, and examining histological changes. Material and MethodsWe used 6-8 week old male and female BALB/c mice. They received 5 {micro}g/g sirolimus via intraperitoneal injections for 5 consecutive days per week for up to 3 weeks (treated group) or DMSO (control group). Upon euthanasia, the gonads were promptly removed while the heart was still beating, weighed, and processed for cellular respiration, ATP measurement, and histological studies. Cellular respiration was measured using a phosphorescence oxygen analyzer and ATP using a bioluminescent assay system. Histology was performed using processed tissue fragments stained with hematoxylin and eosin (H&E). ResultsCellular ATP levels in testicular tissue were higher than ovarian tissue in both groups, but this was not significant (p=0.4 and 0.2). Although the sirolimus group had higher cellular ATP levels, no significant difference was observed in either testicular (p=0.6) or ovarian (p=0.9) tissue. Both DMSO (p=0.01) and sirolimus groups (p=0.008) showed lower cellular respiration in ovarian than testicular tissue. Cellular respiration was lower in sirolimus group than DMSO group in both testicular (p=0.6) and ovarian (p=0.2) tissue. Testicular cellular respiration remained unchanged up to day 11 before declining, while ovarian respiration reduced up to day 11 and remained unchanged. Testicular histopathology showed normal sperm production and normal follicular development in ovarian tissue. ConclusionThe results suggest that the 5 {micro}g/g dose of sirolimus does not cause significant short-term changes in the cellular bioenergetics of the gonads or produce noticeable histopathological changes.

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Narchi, H., Al Bawardi, A., Thachillath, P., Souid, A.-K.. 2023-05-15. Cellular Bioenergetics Effects of Sirolimus on the Gonads of BALB/c Mice. https://doi.org/10.1101/2023.05.15.540776

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein

The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S-glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo. Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly.

biochemistry↗
bioRxiv2026

Snapshots from the Catalytic Landscape of Chalcone Isomerase

Chalcone isomerase (CHI) catalyzes the cyclization of 3-ring scaffolds of flavonoids, a class of plant-based natural products important for nutrition and disease prevention. A persistent question has been whether the enzyme uses dynamics to facilitate conformational rearrangements of substrates within the active site. To help resolve this question, CHI was crystallized with phloretin, a flexible substrate analogue that cannot undergo cyclization. The crystal structure possesses eight protein molecules per asymmetric unit, revealing different active site conformations that accommodate different bound conformers of phloretin. Together, the structural snapshots depict a series of coordinated, dynamic chemical interactions that lower barriers to substrate rearrangements approaching bond formation. Differential scanning fluorimetry combined with mutational analysis and enzyme kinetics further confirm that phloretin binds to the enzyme active site and that it acts as a competitive inhibitor of CHI. Together these findings answer outstanding questions about the flexibility and dynamics of CHI catalysis, information that may be useful for future biosynthetic design and enzyme engineering goals. Overall, this work supports a catalytic model in which the CHI enzyme operates as a dynamic ensemble of structures necessary to facilitate catalytic substrate rearrangements.

biochemistry↗
bioRxiv2026

Structures of pUG-fold RNA bound to DNMT1 reveal a mechanism for RNA-mediated epigenetic regulation

Many chromatin-associated proteins have been found to bind RNA as a means of epigenetic regulation. Specifically, DNA methyltransferase 1 (DNMT1), which maintains cytosine methylation at CpG dinucleotides, is inhibited by RNA at transcribed DNA loci in cells. However, the mechanisms by which RNA binds DNMT1 and inhibits its activity remain unknown. Here, we determine a series of cryogenic electron microscopy (cryo-EM) structures of human DNMT1 bound to pUG-fold RNA, a non-canonical G-quadruplex previously observed to inhibit activity, revealing two distinct RNA-binding modes. The pUG-fold RNA binds the surface of DNMT1 in its autoinhibited conformation across a positively charged surface between the methyltransferase domain and the CXXC domain, and it binds directly in the active site of an open DNMT1 conformation. RNA binding is sterically incompatible with substrate DNA engagement in both states. Our 2.5 [A] structure captures the intricate network of hydrogen bonds and electrostatic interactions between amino acids in the methyltransferase domain and the tetrad layers of pUG-fold RNA. Metadynamics molecular dynamics simulations provide an orthogonal view of the conformational landscape of DNMT1, revealing the two distinct RNA-binding modes. Furthermore, our analysis of published DNMT1 RIP-seq and eCLIP-seq data confirms that DNMT1-interacting RNAs in cells exhibit a strong propensity to form non-canonical G-quadruplex RNA structures. Collectively, our study provides the first structural basis for pUG-fold RNA recognition by a protein and illustrates how cryo-EM and AI-based methods for protein and RNA structure prediction synergize to inform the mechanism of RNA-mediated regulation of DNMT1.

biochemistry↗