A Mutant Form of Prourokinase That Spares Hemostatic Fibrin

Abstract Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

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Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22147566 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7566

Author(s):

Eleonora Stronati ◽

Stefano Biagioni ◽

Mario Fiore ◽

Mauro Giorgi ◽

Giancarlo Poiana ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Neuronal Differentiation ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Nervous System Development ◽

Mutant Form ◽

Wild Type ◽

Fused In Sarcoma ◽

Glial Lineage

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

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Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

eLife ◽

10.7554/elife.02882 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 131

Author(s):

Akash Das ◽

Michael S Brown ◽

Donald D Anderson ◽

Joseph L Goldstein ◽

Arun Radhakrishnan

Keyword(s):

Plasma Membrane ◽

Regulatory Mechanism ◽

Low Density Lipoprotein ◽

Human Fibroblasts ◽

Density Lipoprotein ◽

Cholesterol Homeostasis ◽

Mutant Form ◽

Perfringolysin O ◽

Cholesterol Levels ◽

Plasma Membrane Cholesterol

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PM's PFO-accessible pool and, after a short lag, it also increases the ER's PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation.

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Exon Splice Enhancer Mutation (GH-E32A) Causes Autosomal Dominant Growth Hormone Deficiency

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2007-0857 ◽

2007 ◽

Vol 92 (11) ◽

pp. 4427-4435 ◽

Cited By ~ 21

Author(s):

Vibor Petkovic ◽

Didier Lochmatter ◽

James Turton ◽

Peter E. Clayton ◽

Peter J. Trainer ◽

...

Keyword(s):

Cell Proliferation ◽

Autosomal Dominant ◽

Secretory Granules ◽

Cellular Level ◽

Hormone Deficiency ◽

Mutant Form ◽

Splice Enhancer ◽

Exon Splice ◽

Microscopy Analysis ◽

Derived Data

Abstract Context and Objective: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing. Design, Setting, and Patients: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A→C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level. Interventions and Results: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH. Conclusion: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

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Productivity of the buckwheat mutant form determinate floret cluster

Russian Agricultural Sciences ◽

10.3103/s1068367415020093 ◽

2015 ◽

Vol 41 (2-3) ◽

pp. 128-131

Author(s):

A. N. Fesenko ◽

O. V. Biryukova ◽

O. A. Shipulin

Keyword(s):

Mutant Form

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Familial Parkinson's Disease Mutant E46K α-Synuclein Localizes to Membranous Structures, Forms Aggregates, and Induces Toxicity in Yeast Models

ISRN Neurology ◽

10.5402/2011/521847 ◽

2011 ◽

Vol 2011 ◽

pp. 1-14 ◽

Cited By ~ 4

Author(s):

Michael Fiske ◽

Michael White ◽

Stephanie Valtierra ◽

Sara Herrera ◽

Keith Solvang ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Plasma Membrane ◽

Mutant Form ◽

Phospholipid Membrane ◽

Endomembrane System ◽

Wild Type ◽

Significant Toxicity ◽

Familial Parkinson’S Disease

In Parkinson’s disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.

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Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

Journal of Bacteriology ◽

10.1128/jb.01435-09 ◽

2010 ◽

Vol 192 (13) ◽

pp. 3452-3463 ◽

Cited By ~ 17

Author(s):

Nicole M. Dupes ◽

Brian W. Walsh ◽

Andrew D. Klocko ◽

Justin S. Lenhart ◽

Heather L. Peterson ◽

...

Keyword(s):

Bacillus Subtilis ◽

Dna Replication ◽

Mismatch Repair ◽

Mutation Frequency ◽

Elevated Temperatures ◽

Temperature Sensitive ◽

Mutant Form ◽

Appreciable Effect ◽

Missense Mutations ◽

Dna Mismatch

ABSTRACT The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.

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