scholarly journals Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Farhana Taher Sumya ◽  
Irina D. Pokrovskaya ◽  
Vladimir Lupashin

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.

2021 ◽  
Vol 118 (21) ◽  
pp. e2016904118
Author(s):  
Derek K. Cheng ◽  
Tobiloba E. Oni ◽  
Jennifer S. Thalappillil ◽  
Youngkyu Park ◽  
Hsiu-Chi Ting ◽  
...  

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.


2010 ◽  
Vol 192 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Jyl S. Matson ◽  
Hyun Ju Yoo ◽  
Kristina Hakansson ◽  
Victor J. DiRita

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.


2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Tomohiro Yamasaki ◽  
Adrian Lita ◽  
Tyrone Dowdy ◽  
Mark Gilbert ◽  
Mioara Larion

Abstract BACKGROUND Gliomas with isocitrate dehydrogenase (IDH) mutations in adults evolve from lower-grade gliomas to secondary glioblastomas (GBM), a fatal disease with fast progression. IDH mutation occurs early in tumorigenesis, and persistently contribute to the reprograming of glucose, lipid and amino acid metabolism. This offer a plethora of potential biomarkers of progression. However, because it is extremely difficult to detect the distribution and transfer of metabolites changing in every moment in a single cell, the involvement of metabolites produced by mutant IDH in malignant progression remains understudied. MATERIALS AND METHODS Raman imaging spectroscopy, which can image chemical bonds and concentration of molecules at submicron spatial resolution, enables detection of spatiotemporal changes of metabolomes in live cells. We developed the software called Biomolecular Component Analysis (BCAbox) to deconvolute the recorded raw Raman spectra, leading to detection of unique spectral features of different classes of biomolecules. RESULTS AND CONCLUSIONS We applied Raman imaging spectroscopy to GBM cell lines that were transfected with IDH1 mutant gene. Our results indicated that lipid metabolism has a unique profile in IDH1 mutant gliomas. Subsequent mass spectrometry analysis of extracted organelle revealed the exact classes of lipids altered in the IDH mutant glioma and suggested biomarkers unique to IDH1 mutant. We will report our validation studies of the biomarkers in patient-derived IDH mutant glioma cell lines and patients derived-orthotopic xenograft mouse models with different degrees of aggressiveness and in matched primary versus recurrent gliomas. The results of the present study may provide novel insights into the discovery of metabolic biomarkers for the malignant progression in IDH mutant gliomas.


2020 ◽  
Vol 21 (9) ◽  
pp. 3268
Author(s):  
Eleonora Pavan ◽  
Maximiliano Ormazabal ◽  
Paolo Peruzzo ◽  
Emilio Vaena ◽  
Paula Rozenfeld ◽  
...  

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson’s disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.


2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.


1993 ◽  
Vol 13 (9) ◽  
pp. 5175-5185 ◽  
Author(s):  
M J Evans ◽  
J E Metherall

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 611-611 ◽  
Author(s):  
Teresa Ezponda ◽  
Relja Popovic ◽  
Yupeng Zheng ◽  
Behnam Nabet ◽  
Christine Will ◽  
...  

Abstract Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that alter the levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of MM cases; nevertheless, the epigenetic impact of UTX loss in MM and the mechanisms by which it contributes to this disease remain to be elucidated. To ascertain the biological impact of UTX loss, we used a recently identified isogenic cell line pair: ARP-1 (UTX wild-type) and ARD (UTX null). UTX-null ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX-null ARD cells have increased clonogenicity compared to UTX-wild-type ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to Hs-5 bone marrow stromal cells and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD cells demonstrating this effect is dependent on UTX loss. Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back. Therefore, UTX depletion may alter H3K27me at specific loci, and control the expression of a limited number of genes. To identify the genes and pathways that are altered upon UTX loss, we performed RNA-sequencing (RNA-seq) on the paired MM cell lines and the add-back system. This analysis revealed approximately 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX in the UTX-null ARD cells reversed the expression of approximately 1,400 genes, most of them being upregulated upon reintroduction of UTX. Gene ontology analysis of genes responsive to UTX manipulation identified pathways such as JAK-STAT, cadherin, integrin and Wnt pathways. Many of these pathways are related to cell adhesion properties, correlating with the effects observed in vitro. Some examples of the genes which expression was restored upon UTX add-back are E-cadherin, whose loss has been associated with MM progression; and PTPN6, a negative regulator of the JAK-STAT pathway. Chromatin immunoprecipitation (ChIP) experiments at UTX target genes revealed a decrease in H3K27me3 and a concomitant increase in H3K4me3 upon UTX add-back, correlating with the observed changes in gene expression. As loss of UTX leads to a failure in the removal of H3K27me3, we hypothesized that UTX-null cells may be more dependent on EZH2 to maintain high H3K27me3 levels at specific loci. Treatment of the paired cell lines with the EZH2 inhibitor GSK343 for 7 days significantly decreased the viability of UTX-null ARD cells, but had no effect on the UTX wild-type ARP-1 cells. This effect was not exclusive to these cell lines, as treatment of a panel of UTX wild-type and mutant MM cells corroborated the increased sensitivity in UTX-mutant cells. RNA-seq of ARD cells treated with GSK343 for 7 days identified approximately 2,000 genes with altered expression in response to this drug, most of them being upregulated upon EZH2 inhibition. These genes partially overlapped with the genes that were responsive to UTX add-back, including E-cadherin, suggesting that treatment with EZH2 inhibitors is somewhat similar to UTX add-back. Collectively, this work demonstrates that loss of UTX alters the epigenetic landscape of MM cells, leading to altered expression of a specific set of genes, ultimately benefiting cells through increased proliferation, clonogenicity and adhesion. Moreover, inhibition of EZH2 partially reverses aberrations promoted by UTX loss and may represent a rationale therapy for the treatment of this type of MM. Disclosures No relevant conflicts of interest to declare.


2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.


2014 ◽  
Vol 26 (1) ◽  
pp. 117 ◽  
Author(s):  
J. Chung ◽  
X. Zhang ◽  
B. Colins ◽  
K. Howard ◽  
S. Simpson ◽  
...  

The high mobility group AT-hook 2 (HMGA2) protein has been shown to be a crucial gene for cell growth, proliferation, and apoptosis; HMGA2 is also a strong biological candidate for growth, because mutations in this gene alter body size in mice and humans. Compared with wild-type controls, adult mice lacking HMGA2 are 60% smaller, and adult heterozygous mutants are 20% smaller. In humans, HMGA2 has been associated with adult and childhood height without any other deleterious effect. Additionally, a microdeletion in the HMGA2 gene in a human patient resulted in short stature, with no dysmorphologies and normal puberty. In order to determine the effect of HMGA2 on fetal and adult growth in pigs, a transgenic pig line deficient in HMGA2 expression was generated by gene targeting in fetal fibroblasts (FF). Using a targeting vector carrying a reporter gene, and homology arms specific to HMGA2, heterozygous mutant cell lines were generated. The cell lines were then used to generate 6 heterozygous females by somatic cell nuclear transfer (SCNT). Bodyweights and lengths from snout to base of tail were measured every 2 weeks for a year for mutant (n = 6) and wild-type farm gilts (n = 6). Data were analysed by one-way ANOVA. As in mice, disruption of one allele of the HMGA2 gene resulted in 25% reduction in weight (P < 0.0001) and 14% reduction in length (P < 0.0001). Early in postnatal growth (2 months), weights of mutants were not different than wild-type. However, mutants were 20 to 35% lighter (P < 0.05) during mid stages (6 months) and 25 to 30% (P < 0.0001) in late stages (3 months). The same insertional mutation generated 8 heterozygous male clones by SCNT. In addition, 7 nontransgenic males from the same FF line were generated as SCNT controls. Bodyweights and lengths were measured every 2 weeks for 30 weeks for HMGA2 heterozygous mutants (n = 8), control SCNT (n = 7) and wild-type farm boars (n = 5). The weight curve of boars showed similar pattern as for mutant gilts. At 30-week postnatal stage, mutants were 17% (P < 0.05) and 16% (P < 0.05) lighter in weight compared with littermate and wild-type animals, respectively. We are presently developing homozygous HMGA2 mutant lines. Currently, 3 of 6 heterozygous gilts have been bred with heterozygous boars, with 1 confirmed pregnancy. The expectation is that the homozygous animals will, like mice, be 60% smaller than the wild-type animals. The approach described here will result not only in a valuable large-animal model of dwarfism, but also in a tool to reduce the size of existing transgenic and nontransgenic swine lines. This, in turn, will increase the receptivity of valuable transgenic lines by the biomedical community. Funding for this work was provided by NIH grant R21-OD010553 to JP.


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