Characterizing the function and regulation of YCX1, a new member of the CCX family of exchangers

This Project Grant award from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training Program (CFDA 93.859) provides $378,750.00 to Saint Louis University to conduct research on YCX1, a newly discovered protein involved in calcium signaling. The project aims to:

1. Characterize the biochemical activity and regulation of YCX1, a putative calcium/cation exchanger, and its role in calcium signaling within the endoplasmic reticulum and Golgi.

2. Determine how YCX1 regulates cellular metabolism by facilitating calcium flow from the endoplasmic reticulum to mitochondria.

3. Examine the function of the YCX1 homolog in the pathogenic fungus Cryptococcus neoformans and its potential role in virulence.

The research is expected to enhance the understanding of calcium signaling and its implications for human health and disease. The project will be conducted at Saint Louis University over the award period of September 10, 2024 to August 31, 2027.

Abstract

Calcium ions play critical roles in regulating many biological processes. MIS-regulation of CA2+ signaling can lead to diseases, and the CA2+ channels and exchangers are among the most important drug targets. Proper CA2+ signaling is also vital for the virulence of many pathogenic organisms. Thus, a thorough understanding of the CA2+ signaling is very important for enhancing human health. The objective of this project is to study YCX1, a newly discovered protein required for proper CA2+ signaling in yeast. Disrupting YCX1 results in a decrease in the levels of cytosolic CA2+ and the activation of calcineurin, a reduced chitin content in the cell wall, an increased sensitivity to thermal stress, and an impaired respiratory growth. YCX1 is found in the endoplasmic curriculum (ER) and golgi but how IT works is not known. Phylogenetics analysis suggests YCX1 is a cation/CA2+ exchanger (CCX), the newest and the least understood family of CA2+ exchangers. Our hypothesis is that YCX1 controls CA2+ release from the ER and golgi and regulates cellular metabolism by modulating CA2+ flow from the ER to mitochondria. To achieve our objectives, three specific aims are proposed: aim 1: characterizing YCX1 and its role in proper CA2+ signaling. To characterize YCX1, we will first determine its biochemical activity by expressing the protein in e. Coli and examine its ability to transport CA2+. To test a role of YCX1 in regulating CA2+ release from the ER and golgi, we will use genetically encoded and organelle specific CA2+ indicators. To determine how YCX1 itself is regulated, we will characterize known YCX1 phosphorylation sites, identify proteins that interact with YCX1, and examine their role in regulating YCX1 and CA2+ signaling. Aim 2: determining the mechanisms by which YCX1 regulates cellular metabolism. CA2+ is required for the activity of pyruvate dehydrogenase, and mitochondria and the ER are tethered in close proximity. We hypothesize YCX1 facilitates CA2+ flow from the ER to mitochondria to aid in the enzyme activation. To test this, we will determine if YCX1 is required for mitochondrial CA2+ and pyruvate dehydrogenase activation during respiratory growth. We will also determine if the tethering of mitochondria and the ER is crucial for the action of YCX1 in regulating respiratory growth and CA2+. Aim 3: characterizing the YCX1 homolog in cryptococcus. C. Neoformans is a significant pathogen responsible for over 180,000 annual deaths. Through bioinformatics analysis, we identified a YCX1 homolog in cryptococcus. This protein may have similar functions to YCX1 and could represent a novel factor crucial for the virulence of cryptococcus. To assess this, we will examine the effects of disrupting this homolog in cryptococcus on calcineurin signaling, chitin level, and sensitivity to 37°c. We will also examine the subcellular localization of this protein and its transporter activity.