Biology subjects

Goncalves, M. M.

Publications and source records attributed to Goncalves, M. M..

3 recordsLinked to original sources

bioRxiv2025

Characterization of conformational dynamics and allostery of the catalytic domain of human mitochondrial YME1L protease

Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L protease is an important contributor to proteostasis which belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protease family and is anchored to the inner mitochondrial membrane. YME1L plays a pivotal role in mitochondrial protein quality control by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1Ls conformational dynamics, proteolytic activity, and stability remain unclear. Here we characterize the conformational dynamics and allosteric regulation of the YME1L catalytic domain. Using a hexameric soluble YME1L construct, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy to demonstrate that nucleotide binding reduces the backbone flexibility and modulates the side-chain dynamics of the AAA+ domain, while Zn{superscript 2} binding stabilizes the protease domain. We also reveal novel long-range allostery between the AAA+ and protease domains of YME1L, mediated by a critical salt bridge on the inter-domain interface. We show the importance of the salt bridge in facilitating ATP-dependent substrate degradation by YME1L. Additionally, we show that ATP binding stabilizes the structure of the catalytic domain of YME1L and protects it from chemical modification- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide novel insights into understanding its proteolytic activity and structural stability under physiological and stress conditions.

biophysics↗

bioRxiv2024

Mechanism of allosteric activation in human mitochondrial ClpP protease

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement. Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.

biochemistry↗

bioRxiv2021

Evaluation of the efficacy of simethicone in the abdominal ultrasound and pancreas evaluation on dogs

Intestinal gas results in low quality imaging in abdominal ultrasound in domestic animals. On dogs, suggested preparation protocols are varied and low studied. The aim of this work was comparing the efficacy between two preparations in improving the complete abdominal ultrasound exam and pancreas preview. 40 dogs were enrolled in this study. They were separated in two groups (NS diet: fasting; WS diet: fasting added to simethicone). The pancreas images were taken from left lateral decubitus. A score of one to three (1: bad, 2: fair and 3: good) measured separately the quality of the complete exam, left and right limbs and pancreatic body. In both treatments, there were results above 50% of good images of the complete exam and right limb of the pancreas. Otherwise, there were results above 90% of bad images of the right limb and pancreatic body. According to the fact of there is no statistically significant difference between the diets, as well submitted animals to the NS diet as the WS diet, all the results obtained from this work (bad or good images) could be acquired from any of both diets. The good abdominal exam preview as well submitted animals to the simethicone as those without the medication refute this medicine requirement to the abdominal ultrasound exam, but not to the evaluation of the left limb and pancreatic body.

pharmacology and toxicology↗