scholarly journals VCP protects neurons from proteopathic seeding

2021 ◽  
Author(s):  
Jiang Zhu ◽  
Sara Pittman ◽  
Dhruva Dhavale ◽  
Rachel French ◽  
Jessica N Patterson ◽  
...  
Keyword(s):  
Neuronal Degeneration ◽  
Data Support ◽  
Genome Wide ◽  
Disease Mutations ◽  
A Genome ◽  
Cellular Machinery ◽  
Seeding Efficiency ◽  
Intrastriatal Injection ◽  
In Cells

Uptake and spread of proteopathic seeds, such as αS, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase αS seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of αS seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to αS since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

scholarly journals Genome-Wide Analysis of Chromosomal Features Repressing Human Immunodeficiency Virus Transcription

2005 ◽  
Vol 79 (11) ◽  
pp. 6610-6619 ◽  
Author(s):  
M. K. Lewinski ◽  
D. Bisgrove ◽  
P. Shinn ◽  
H. Chen ◽  
C. Hoffmann ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Fluorescent Protein ◽  
Centromeric Heterochromatin ◽  
Regulatory Sequences ◽  
Hiv Transcription ◽  
Genome Wide ◽  
A Genome ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
In Cells

ABSTRACT We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

scholarly journals Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Spencer Hill ◽  
Kurt Reichermeier ◽  
Daniel C Scott ◽  
Lorena Samentar ◽  
Jasmin Coulombe-Huntington ◽  
...  
Keyword(s):  
Chain Extension ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Extension Activity ◽  
The Stability ◽  
Genetic Buffering ◽  
Ligase Function ◽  
In Cells

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

SERP1 is an assembly regulator of γ-secretase in metabolic stress conditions

2020 ◽  
Vol 13 (623) ◽  
pp. eaax8949 ◽  
Author(s):  
Sunmin Jung ◽  
Junho Hyun ◽  
Jihoon Nah ◽  
Jonghee Han ◽  
Seo-Hyun Kim ◽  
...  
Keyword(s):  
Er Stress ◽  
Parietal Lobe ◽  
Metabolic Stress ◽  
Expression Library ◽  
Genome Wide ◽  
Secretase Activity ◽  
A Genome ◽  
Aβ Generation ◽  
Aβ Production ◽  
In Cells

The enzyme γ-secretase generates β-amyloid (Aβ) peptides by cleaving amyloid protein precursor (APP); the aggregation of these peptides is associated with Alzheimer’s disease (AD). Despite the development of various γ-secretase regulators, their clinical use is limited by coincident disruption of other γ-secretase–regulated substrates, such as Notch. Using a genome-wide functional screen of γ-secretase activity in cells and a complementary DNA expression library, we found that SERP1 is a previously unknown γ-secretase activator that stimulates Aβ generation in cells experiencing endoplasmic reticulum (ER) stress, such as is seen with diabetes. SERP1 interacted with a subcomplex of γ-secretase (APH1A/NCT) through its carboxyl terminus to enhance the assembly and, consequently, the activity of the γ-secretase holoenzyme complex. In response to ER stress, SERP1 preferentially recruited APP rather than Notch into the γ-secretase complex and enhanced the subcellular localization of the complex into lipid rafts, increasing Aβ production. Moreover, SERP1 abundance, γ-secretase assembly, and Aβ production were increased both in cells exposed to high amounts of glucose and in diabetic AD model mice. Conversely, Aβ production was decreased by knocking down SERP1 in cells or in the hippocampi of mice. Compared to postmortem samples from control individuals, those from patients with AD showed increased SERP1 expression in the hippocampus and parietal lobe. Together, our findings suggest that SERP1 is an APP-biased regulator of γ-secretase function in the context of cell stress, providing a possible molecular explanation for the link between diabetes and sporadic AD.

scholarly journals VCP Protects Neurons from Proteopathic Seeding

2021 ◽  
Author(s):  
Jiang Zhu ◽  
Sara Pittman ◽  
Dhruva Dhavale ◽  
Rachel French ◽  
Jessica N. Patterson ◽  
...  
Keyword(s):  
Alpha Synuclein ◽  
Neuronal Uptake ◽  
Primary Neurons ◽  
Guide Rna ◽  
Late Endosomes ◽  
A Genome ◽  
Cellular Machinery ◽  
Or Gene ◽  
Intrastriatal Injection

Abstract Background: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), tau, and TDP-43, contribute to neurodegeneration and disease progression. The cellular machinery necessary for this process is poorly understood. Methods: Cas9 expressing αS FRET biosensors were transduced with a whole-genome guide RNA (gRNA) library, seeded with αS fibrils, and flow-sorted. Candidate genes protective against αS seeding were identified following gRNA sequencing of FRET+ and FRET- cell populations. Secondary validation of the high probability candidate suppressor VCP, utilized VCP inhibitors or gene knockdown in αS biosensors and primary neurons. In vivo validation was performed in VCP disease mutation mice following intrastriatal injection of αS seeds. TDP-43 seeding was performed in primary neurons from control or VCP mutant mice.Results: We devised a genome-wide CRISPR-Cas9 screen to identify suppressors of αS seeding. This approach identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) a phenotypically and pathologically variable neurodegeneraive disease characterized by myopathy, motor neuron disease and dementia with TDP-43, αS and tau inclusions. VCP inhibition or MSP disease mutations increased αS seeding in cells and primary cultured neurons. This was similar to treatment with the lysosomal damaging agent, LLoMe or knockdown of the endolysosomal damage response associated VCP cofactor, UBXD1. Intrastriatal injection of αS seeds into VCP disease mice demonstrated enhanced seeding efficiency as compared with controls. Finally, this phenomenon was not specific to αS since VCP disease mutant expression increased TDP-43 seeding in neurons.Conclusion: VCP surveillance of permeabilized late endosomes protects neurons against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

scholarly journals R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ramin Dubey ◽  
Peter van Kerkhof ◽  
Ingrid Jordens ◽  
Tomas Malinauskas ◽  
Ganesh V Pusapati ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Wnt Signaling ◽  
Cultured Cells ◽  
Heparan Sulfate Proteoglycans ◽  
G Protein Coupled Receptors ◽  
Binding Domains ◽  
Genome Wide ◽  
A Genome ◽  
G Protein Coupled ◽  
In Cells

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

A genome-wide linkage scan for familial partial lipodystrophy susceptibility genes in a German kindreds

2007 ◽  
Vol 30 (4) ◽  
pp. 86
Author(s):  
M. Lanktree ◽  
J. Robinson ◽  
J. Creider ◽  
H. Cao ◽  
D. Carter ◽  
...  
Keyword(s):  
Subcutaneous Fat ◽  
Visceral Obesity ◽  
Fat Distribution ◽  
Dominant Negative ◽  
Molecular Forms ◽  
Partial Lipodystrophy ◽  
Genome Wide ◽  
A Genome ◽  
Familial Partial Lipodystrophy ◽  
Promoter Mutations

Background: In Dunnigan-type familial partial lipodystrophy (FPLD) patients are born with normal fat distribution, but subcutaneous fat from extremities and gluteal regions are lost during puberty. The abnormal fat distribution leads to the development of metabolic syndrome (MetS), a cluster of phenotypes including hyperglycemia, dyslipidemia, hypertension, and visceral obesity. The study of FPLD as a monogenic model of MetS may uncover genetic risk factors of the common MetS which affects ~30% of adult North Americans. Two molecular forms of FPLD have been identified including FPLD2, resulting from heterozygous mutations in the LMNA gene, and FPLD3, resulting from both heterozygous dominant negative and haploinsufficiency mutations in the PPARG gene. However, many patients with clinically diagnosed FPLD have no mutation in either LMNA or PPARG, suggesting the involvement of additional genes in FPLD etiology. Methods: Here, we report the results of an Affymetrix 10K GeneChip microarray genome-wide linkage analysis study of a German kindred displaying the FPLD phenotype and no known lipodystrophy-causing mutations. Results: The investigation identified three chromosomal loci, namely 1q, 3p, and 9q, with non-parametric logarithm of odds (NPL) scores >2.7. While not meeting the criteria for genome-wide significance, it is interesting to note that the 1q and 3p peaks contain the LMNA and PPARG genes respectively. Conclusions: Three possible conclusions can be drawn from these results: 1) the peaks identified are spurious findings, 2) additional genes physically close to LMNA, PPARG, or within 9q, are involved in FPLD etiology, or 3) alternative disease causing mechanisms not identified by standard exon sequencing approaches, such as promoter mutations, alternative splicing, or epigenetics, are also responsible for FPLD.

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