A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression

Glioblastoma (GBM) is the most frequent and inevitably lethal primary brain cancer in adults. It is recognized that the overexpression of the endosomal Na+/H+ exchanger NHE9 is a potent driver of GBM progression. Patients with NHE9 overexpression have a threefold lower median survival relative to GBM patients with normal NHE9 expression, using available treatment options. New treatment strategies tailored for this GBM subset are much needed. According to the prevailing model, NHE9 overexpression leads to an increase in plasma membrane density of epidermal growth factor receptors (EGFRs) which consequently enhances GBM cell proliferation and migration. However, this increase is not specific to EGFRs. In fact, the hallmark of NHE9 overexpression is a pan‐specific increase in plasma membrane receptors. Paradoxically, we report that this gain of function in NHE9 can be exploited to effectively target GBM cells for destruction. When exposed to gold nanoparticles, NHE9 overexpressing GBM cells accumulated drastically high amounts of gold via receptor‐mediated endocytosis, relative to control. Irradiation of these cells with near‐infrared light led to apoptotic tumour cell death. A major limitation for delivering therapeutics to GBM cells is the blood‐brain barrier (BBB). Here, we demonstrate that macrophages loaded with gold nanoparticles can cross the BBB, deliver the gold nanoparticles and effect the demise of GBM cells. In combination with receptor tyrosine kinase inhibition, we show this approach holds great promise for a new GBM‐targeted therapy.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152030/1/jcmm14665.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152030/2/jcmm14665_am.pd

The Structure and Function of Frataxin

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich’s ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin’s specific function remains a point of controversy, a general consensus is the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein’s role in different cellular pathways

MicroRNA-135a regulates NHE9 to inhibit proliferation and migration of glioblastoma cells

Abstract Background Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified. Methods Bioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation. Results We show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane. Conclusions We propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.https://deepblue.lib.umich.edu/bitstream/2027.42/140393/1/12964_2017_Article_209.pd

NMR Assignments of a Stable Processing Intermediate of Human Frataxin

Frataxin, a nuclear encoded protein targeted to the mitochondrial matrix, has recently been implicated as an iron chaperone that delivers ferrous iron to the iron-sulfur assembly enzyme IscU. During transport across the mitochondrial membrane, the N-terminal mitochondrial targeting sequence of frataxin is cleaved in a two-step process to produce the mature protein found in the matrix, however N-terminal extended forms of the protein have also been observed in vivo. The recent structural characterization studies of the human frataxin ortholog were performed on a truncated variant of the protein. Here we report the NMR spectral assignment of an extended form of the mature human frataxin ortholog as the basis for understanding the role of the N-terminal domain in protein function

The Metal Centers of Particulate Methane Monooxygenase fromMethylosinus trichosporiumOB3b†‡

This research paper will develop a regression model and analyze the relationship between the dependent variable, housing prices, and the explanatory variables: crime levels, education, income, income inequality, and racial/ethnic population. Data will be collected using metropolitan areas in the United States over a five year span. The dependent variable represents the median house price in a metropolitan area per year and will be measured in thousands of dollars. Crime levels will be measured as two distinct variables: violent crimes and property crimes. Both will be measured in terms of the rate of crime per hundred-thousandth person per year in a given metropolitan area. Education represents the total funding a metropolitan area gets resourced per student per year. Income represents the median household income within a metropolitan area over median household size per year measured in thousands of dollars. Income inequality represents the gap between household income measured as a value between 0 and 1, the gini index, in a metropolitan area per year. Racial/ ethnic makeup is broken down into Hispanic, Asian/ Pacific Islander, African American, Native American, and White or European descent, all of which will be measured as a percentage of population in a metropolitan area per year. It is expected that the estimated coefficient for crime levels, racial/ethnic population, and income inequality will be negative and for education and income, the sign will be positive. The regression analysis will interpret the coefficients and test for the strength of influence using a separate equation; furthermore, important components of the regression will also be explored. The goal of this regression is to understand how the explanatory variables influence the changes that occur in house prices, how they are related to each other, and the how income inequality plays a role