IU Indianapolis ScholarWorks :: Browsing by Subject "Biochemistry "

Browsing by Subject "Biochemistry"

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A same day α-synuclein RT-QuIC seed amplification assay for synucleinopathy biospecimens
(Springer Nature, 2025) Parveen, Sabiha; Alam, Parvez; Orrù, Christina D.; Vascellari, Sarah; Hughson, Andrew G.; Zou, Wen-Quan; Beach, Thomas G.; Serrano, Geidy E.; Goldstein, David S.; Ghetti, Bernardino; Cossu, Giovanni; Pisano, Giada; Pinna, Beatrice; Caughey, Byron; Pathology and Laboratory Medicine, School of Medicine
Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and other synucleinopathies are characterized by the accumulation of abnormal, self-propagating aggregates of α-synuclein. RT-QuIC or seed amplification assays are currently showing unprecedented diagnostic sensitivities and specificities for synucleinopathies even in prodromal phases years in advance of the onset of Parkinsonian signs or dementia. However, commonly used α-synuclein seed amplification assays take ≥48 h to perform as applied to patients’ diagnostic biospecimens. Here, we report the development of a faster α-synuclein RT-QuIC assay that is as analytically sensitive as prior assays of this type, but can be completed in ≤12 h for brain, skin, and intestinal mucosa, with positive signals often arising in <5 h. CSF assays took a few hours longer. Our same-day α-synuclein RT-QuIC (sdRT-QuIC) assay should increase the practicality, cost-effectiveness, and throughput of measurements of pathological forms of α-synuclein for fundamental research, clinical diagnosis, and therapeutics development.
ADAR3 activates NF-κB signaling and promotes glioblastoma cell resistance to temozolomide
(Springer Nature, 2022-08-03) Kurup, Reshma Raghava; Oakes, Eimile K.; Vadlamani, Pranathi; Nwosu, Obi; Danthi, Pranav; Hundley, Heather A.; Medicine, School of Medicine
The RNA binding protein ADAR3 is expressed exclusively in the brain and reported to have elevated expression in tumors of patients suffering from glioblastoma compared to adjacent brain tissue. Yet, other studies have indicated that glioblastoma tumors exhibit hemizygous deletions of the genomic region encompassing ADAR3 (10p15.3). As the molecular and cellular consequences of altered ADAR3 expression are largely unknown, here we directly examined the impacts of elevated ADAR3 in a glioblastoma cell line model. Transcriptome-wide sequencing revealed 641 differentially expressed genes between control and ADAR3-expressing U87-MG glioblastoma cells. A vast majority of these genes belong to pathways involved in glioblastoma progression and are regulated by NF-κB signaling. Biochemical and molecular analysis indicated that ADAR3-expressing U87-MG cells exhibit increased NF-κB activation, and treatment with an NF-κB inhibitor abrogated the impacts of ADAR3 on gene expression. Similarly, we found that increased cell survival of ADAR3-expressing cells to temozolomide, the preferred chemotherapeutic for glioblastoma, was due to increased NF-κB activity. Aberrant constitutive NF-κB activation is a common event in glioblastoma and can impact both tumor progression and resistance to treatment. Our results suggest that elevated ADAR3 promotes NF-κB activation and a gene expression program that provides a growth advantage to glioblastoma cells.
AUTHOR CORRECTION: Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration
(Springer Nature, 2021-08-03) Zhou, Libin; Nho, Kwangsik; Haddad, Maria G.; Cherepacha, Nicole; Tubeleviciute‑Aydin, Agne; Tsai, Andy P.; Saykin, Andrew J.; Sjöström, P. Jesper; LeBlanc, Andrea C.; Radiology and Imaging Sciences, School of Medicine
C. elegans germ granules require both assembly and localized regulators for mRNA repression
(Springer Nature, 2021-02-12) Aoki, Scott Takeo; Lynch, Tina R.; Crittenden, Sarah L.; Bingman, Craig A.; Wickens, Marvin; Kimble, Judith; Biochemistry and Molecular Biology, School of Medicine
Cytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.
Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium
(Springer Nature, 2023-05-21) Ning, Ke; Bhuckory, Mohajeet B.; Lo, Chien‑Hui; Sendayen, Brent E.; Kowal, Tia J.; Chen, Ming; Bansal, Ruchi; Chang, Kun‑Che; Vollrath, Douglas; Berbari, Nicolas F.; Mahajan, Vinit B.; Hu, Yang; Sun, Yang; Biology, School of Science
Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.
Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine
(Springer Nature, 2021-03-22) Kočar, Eva; Lenarčič, Tea; Hodnik, Vesna; Panevska, Anastasija; Huang, Yunjie; Bajc, Gregor; Kostanjšek, Rok; Naren, Anjaparavanda P.; Maček, Peter; Anderluh, Gregor; Sepčić, Kristina; Podobnik, Marjetka; Butala, Matej; Pediatrics, School of Medicine
Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.
Effects of microRNA-298 on APP and BACE1 translation differ according to cell type and 3′-UTR variation
(Springer, 2022-02-23) Wang, Ruizhi; Lahiri, Debomoy K.; Psychiatry, School of Medicine
Alzheimer’s disease (AD) is marked by neurofibrillary tangles and senile plaques composed of amyloid β (Aβ) peptides. However, specific contributions of different cell types to Aβ deposition remain unknown. Non-coding microRNAs (miRNA) play important roles in AD by regulating translation of major associated proteins, such as Aβ precursor protein (APP) and β-site APP-cleaving enzyme (BACE1), two key proteins associated with Aβ biogenesis. MiRNAs typically silence protein expression via binding specific sites in mRNAs’ 3′-untranslated regions (3′-UTR). MiRNAs regulate protein levels in a cell-type specific manner; however, mechanisms of the variation of miRNA activity remain unknown. We report that miR-298 treatment reduced native APP and BACE1 protein levels in an astrocytic but not in a neuron-like cell line. From miR-298’s effects on APP-3′-UTR activity and native protein levels, we infer that differences in APP 3′-UTR length could explain differential miR-298 activity. Such varied or truncated, but natural, 3′-UTR specific to a given cell type provides an opportunity to regulate native protein levels by particular miRNA. Thus, miRNA’s effect tailoring to a specific cell type, bypassing another undesired cell type with a truncated 3′-UTR would potentially advance clinically-relevant translational research.
Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach
(JOVE, 2016-12-17) Panfair, Dilrajkaur; Kusmierczyk, Andrew R.; Biology, School of Science
Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.
Genetic modulation of protein expression in rat brain
(Elsevier, 2025-02-21) Li, Ling; Wu, Zhiping; Guarracino, Andrea; Villani, Flavia; Kong, Dehui; Mancieri, Ariana; Zhang, Aijun; Saba, Laura; Chen, Hao; Brozka, Hana; Vales, Karel; Senko, Anna N.; Kempermann, Gerd; Stuchlik, Ales; Pravenec, Michal; Lechner, Joseph; Prins, Pjotr; Mathur, Ramkumar; Lu, Lu; Yang, Kai; Peng, Junmin; Williams, Robert W.; Wang, Xusheng; Pediatrics, School of Medicine
Genetic variations in protein expression are implicated in a broad spectrum of common diseases and complex traits but remain less explored compared to mRNA and classical phenotypes. This study systematically analyzed brain proteomes in a rat family using tandem mass tag (TMT)-based quantitative mass spectrometry. We quantified 8,119 proteins across two parental strains (SHR/Olalpcv and BN-Lx/Cub) and 29 HXB/BXH recombinant inbred (RI) strains, identifying 597 proteins with differential expression and 464 proteins linked to cis-acting quantitative trait loci (pQTLs). Proteogenomics identified 95 variant peptides, and sex-specific analyses revealed both shared and distinct cis-pQTLs. We improved the ability to pinpoint candidate genes underlying pQTLs by utilizing the rat pangenome and explored the connections between pQTLs in rats and human disorders. Collectively, this study highlights the value of large proteo-genetic datasets in elucidating protein modulation in the brain and its links to complex central nervous system (CNS) traits.
Genome-wide association study of brain biochemical phenotypes reveals distinct genetic architecture of Alzheimer's disease related proteins
(BMC, 2023-01-07) Oatman, Stephanie R.; Reddy, Joseph S.; Quicksall, Zachary; Carrasquillo, Minerva M.; Wang, Xue; Liu, Chia‑Chen; Yamazaki, Yu; Nguyen, Thuy T.; Malphrus, Kimberly; Heckman, Michael; Biswas, Kristi; Nho, Kwangsik; Baker, Matthew; Martens, Yuka A.; Zhao, Na; Kim, Jun Pyo; Risacher, Shannon L.; Rademakers, Rosa; Saykin, Andrew J.; DeTure, Michael; Murray, Melissa E.; Kanekiyo, Takahisa; Alzheimer’s Disease Neuroimaging Initiative; Dickson, Dennis W.; Bu, Guojun; Allen, Mariet; Ertekin‑Taner, Nilüfer; Radiology and Imaging Sciences, School of Medicine
Background: Alzheimer's disease (AD) is neuropathologically characterized by amyloid-beta (Aβ) plaques and neurofibrillary tangles. The main protein components of these hallmarks include Aβ40, Aβ42, tau, phosphor-tau, and APOE. We hypothesize that genetic variants influence the levels and solubility of these AD-related proteins in the brain; identifying these may provide key insights into disease pathogenesis. Methods: Genome-wide genotypes were collected from 441 AD cases, imputed to the haplotype reference consortium (HRC) panel, and filtered for quality and frequency. Temporal cortex levels of five AD-related proteins from three fractions, buffer-soluble (TBS), detergent-soluble (Triton-X = TX), and insoluble (Formic acid = FA), were available for these same individuals. Variants were tested for association with each quantitative biochemical measure using linear regression, and GSA-SNP2 was used to identify enriched Gene Ontology (GO) terms. Implicated variants and genes were further assessed for association with other relevant variables. Results: We identified genome-wide significant associations at seven novel loci and the APOE locus. Genes and variants at these loci also associate with multiple AD-related measures, regulate gene expression, have cell-type specific enrichment, and roles in brain health and other neuropsychiatric diseases. Pathway analysis identified significant enrichment of shared and distinct biological pathways. Conclusions: Although all biochemical measures tested reflect proteins core to AD pathology, our results strongly suggest that each have unique genetic architecture and biological pathways that influence their specific biochemical states in the brain. Our novel approach of deep brain biochemical endophenotype GWAS has implications for pathophysiology of proteostasis in AD that can guide therapeutic discovery efforts focused on these proteins.

Browsing by Subject "Biochemistry"

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