Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

Abstract

Abbreviations: ALADIN, alacrima-achalasia-adrenal insufficiency neurologic disorder; CYP, cytochrome P450; ER, endoplasmic reticulum; GFP, green fluorescent protein; NADPH, Nicotinamide adenine dinucleotide phosphate; NUP, nucleoporin; PGRMC1 and 2, progesterone receptor membrane component 1 and 2 Investigating cell division in human adrenal cells we show that proliferation is decreased upon overexpression of ALADIN, PGRMC1 or PGRMC2.• In immunofluorescence experiments using human adrenal cells and triple A patient fibroblasts we observed that during cell division PGRMC1 localizes to the microtubule kinetochore-fibers in metaphase and to the mid-body in telophase.• Depletion of ALADIN results in mis-localization of Aurora A and PGRMC1 in metaphase cells of the human adrenal cell line and fibroblasts derived from patients with triple A syndrome.• In real time PCR using RNA of fibroblasts of triple A syndrome patients and healthy controls we measured an increased expression of PGRMC2 in cells with ALADIN mis-function compared to the control cells.• We hypothesize that a loss of the regulatory interaction between ALADIN and PGRMC2 leads to an over-regulation and over-expression of PGRMC2 and displaces PGRMC1 at the metaphase spindle.This diminishing of PGRMC1 concentration at kinetochore fibers may lead to mitotic errors and proliferation arrest. ABSTRACTMembrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxification. PGRMC1 and PGRMC2 belong to this group of microsomal receptors and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other thereby suppressing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic controller Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over-expressed. Furthermore, we find that depletion of ALADIN results in mis-localization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fibroblasts. Our results emphasize the possibility that loss of the regulatory interaction between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibers and to mitotic errors. This observation may explain part of the symptoms seen in triple A syndrome patients.Cells were seeded in 24-well culture dishes at a density of 0.4 x10 5 cells/well 24 h before proliferation analysis. Confluence measurement was done using live cell imaging on IncuCyte Zoom (Essen BioScience, Ann Arbor MI, USA) over at least 65 h. Measurement was done at least in triplicate and experiments were repeated at least twice. Immunofluorescence microscopyCells grown on g...

PGRMC1 phosphorylation and cell plasticity 2: genomic integrity and CpG methylation

“…We show that changes in posttranslational modification of PGRMC1 are involved in the maintenance of genomic integrity. Future research should examine whether this involves previously reported PGRMC1 involvement with G0/G1 checkpoint and/or spindle association (Cahill et al, 2016a;Griffin et al, 2014;Juhlen et al, 2018;Luciano and Peluso, 2016;Terzaghi et al, 2016).…”

“…Progesterone (P4) Receptor Membrane Components 1 (PGRMC1) is a cytochrome b5 (cytb5) protein and a member of the membrane-associated P4 receptor (MAPR) family with a plethora of cellular functions. These include some cytb5-typical functions that predate or are potentially ancient in eukaryotes (regulation of heme synthesis (Piel et al, 2016), cyP450 (cytochrome P450) interactions (Oda et al, 2011;Ryu et al, 2017;Szczesna-Skorupa and Kemper, 2011), sterol metabolism (Cahill and Medlock, 2017;Hughes et al, 2007;Mallory et al, 2005)), some that evidently arose in eukaryotes (membrane trafficking (reviewed by: Cahill et al, 2016a;Mifsud and Bateman, 2002), cell cycle regulation at the G0/G1 checkpoint (Cahill et al, 2016a;Griffin et al, 2014;, mitotic/meiotic spindle association (Juhlen et al, 2018;Luciano and Peluso, 2016;Terzaghi et al, 2016)), and clearly specialized metazoan functions including e.g., fertility, embryogenic axon guidance, and membrane trafficking associated with synaptic plasticity (reviewed by: Cahill et al, 2016a;Rohe et al, 2009). CyP450-interactions (Ryu et al, 2017) conspicuously feature PGRMC1 regulation of the most conserved eukaryotic cyP450 (lanosterol 14-alpha demethylase, CYP51A) to modify the first sterol (lanosterol) from yeast to mammals (Cahill, 2007;Hughes et al, 2007;Mallory et al, 2005).…”

PGRMC1 effects on metabolism, genomic mutation and CpG methylation imply crucial roles in animal biology and disease

“…Our data shows that changes in posttranslational modification of PGRMC1 result in altered metabolism, genomic mutation rates, and epigenetic methylation status. Future research should examine whether genomic effects involve previously reported PGRMC1 involvement with G0/G1 checkpoint and/or spindle association [2,[20][21][22][23][24].…”

“…Some functions evidently arose in eukaryotes (e.g. membrane trafficking (reviewed by: [2,19]), cell cycle regulation at the G0/G1 checkpoint [2,20,21], mitotic/meiotic spindle association [22][23][24]). Other clearly specialized metazoan functions include e.g., hormonal influence on fertility, embryogenic axon guidance, and membrane trafficking associated with synaptic plasticity [reviewed by : 2, 25].…”