The extracellular β-glucosidase BGL2 has two variants with different molecular sizes and hydrolytic activities in the stipe or pilei of Coprinopsis cinerea

Microbiology ◽  
2021 ◽  
Vol 167 (11) ◽  
Author(s):  
Rujuan Dai ◽  
Mingmei Yang ◽  
Jing Zhao ◽  
Xiao Liu ◽  
Yajun Zhou ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Rna Splicing ◽  
Splice Variants ◽  
Proteolytic Cleavage ◽  
Apparent Molecular Mass ◽  
Full Length ◽  
Coprinopsis Cinerea ◽  
Terminal Fragment ◽  
Hydrolytic Activities

Two variants of extracellular β-glucosidase (BGL2) were purified from the stipe and pilei of Coprinopsis cinerea. In the stipe, BGL2 was a monomeric protein with an apparent molecular mass of approximately 220 kDa, representing a mature full-length peptide of BGL2. However, in the pilei, the apparent molecular mass of BGL2 was only approximately 120 kDa, consisting of the 60 kDa N-terminal fragment and 55 kDa C-terminal fragment. The hydrolytic activities of BGL2 purified from the pilei were higher than those of BGL2 purified from the stipe. No mRNA splice variants of bgl2 were detected. Therefore, the different variants of BGL2 in the stipe and pilei were not formed by differential RNA splicing. Furthermore, in vitro experiments showed that full-length BGL2 could be cleaved by endogenous proteases from pilei or commercial trypsin at a similar site to form an oligomeric protein consisting of the N-terminal fragment and C-terminal fragment similar to BGL2 from pilei. The hydrolytic activity of BGL2 increased after cleavage by those proteases in vitro. We conclude that the 120 kDa variant of BGL2 in the pilei of C. cinerea is formed by posttranslational proteolytic cleavage. Posttranslational proteolytic cleavage is an efficient way to regulate the activity of BGL2 to adapt to the needs of different physiological functions in the elongation stipe and expansion pilei of C. cinerea.

scholarly journals A short ERK5 isoform modulates nucleocytoplasmic shuttling of active ERK5 and associates with poor survival in breast cancer

2021 ◽  
Author(s):  
Mariska Miranda ◽  
Jodi M. Saunus ◽  
Seçkin Akgül ◽  
Mahdi Moradi Marjaneh ◽  
Jamie R. Kutasovic ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Splice Variants ◽  
Clinicopathological Features ◽  
Full Length ◽  
Nucleocytoplasmic Shuttling ◽  
Poor Survival ◽  
Mesenchymal Transition

AbstractBackgroundThe nucleocytoplasmic shuttling of ERK5 has gained recent attention as a regulator of its diverse roles in cancer progression but the exact mechanisms for this shuttling are still under investigation.MethodsUsing in vitro, in vivo and in silico studies, we investigated the roles of shorter ERK5 isoforms in regulating the nucleocytoplasmic shuttling of active phosphorylated-ERK5 (pERK5). Retrospective cohorts of primary and metastatic breast cancer cases were used to evaluate the association of the subcellular localization of pERK5 with clinicopathological features.ResultsExtranuclear localization of pERK5 was observed during cell migration in vitro and at the invasive fronts of metastatic tumors in vivo. The nuclear and extranuclear cell fractions contained different isoforms of pERK5, which are encoded by splice variants expressed in breast and other cancers in the TCGA data. One isoform, isoform-3, lacks the C-terminal transcriptional domain and the nuclear localization signal. The co-expression of isoform-3 and full-length ERK5 associated with high epithelial-to-mesenchymal transition (EMT) and poor patient survival. Experimentally, expressing isoform-3 with full-length ERK5 in breast cancer cells increased cell migration, drove EMT and led to tamoxifen resistance. In breast cancer patient samples, pERK5 showed variable subcellular localizations where its extranuclear localization associated with aggressive clinicopathological features, metastasis, and poor survival.ConclusionOur studies support a model of ERK5 nucleocytoplasmic shuttling driven by splice variants in an interplay between mesenchymal and epithelial states during metastasis. Using ERK5 as a biomarker and a therapeutic target should account for its splicing and context-dependent biological functions.Graphical AbstractERK5 isoform-3 expression deploys active ERK5 (pERK5) outside the nucleus to facilitate EMT and cell migration. In cells dominantly expressing isoform-1, pERK5 shuttles to the nucleus to drive cell expansion.

scholarly journals Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

2000 ◽  
Vol 345 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Bruno ANTONSSON ◽  
Sylvie MONTESSUIT ◽  
Sandra LAUPER ◽  
Robert ESKES ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Molecular Mass ◽  
Lipid Membranes ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Cytochrome C Release ◽  
Octyl Glucoside ◽  
Apoptotic Activity

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Abstract Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

Proteolytic Modulation of Thrombopoietin Activity: Comparison of Thrombin, Plasmin, and Urokinase

2000 ◽  
Vol 83 (06) ◽  
pp. 909-914 ◽  
Author(s):  
Kaelen Aramaki ◽  
Alexander Reiner
Keyword(s):  
Marked Decrease ◽  
Limited Proteolysis ◽  
Full Length ◽  
Time Dependent ◽  
Dependent Manner ◽  
Terminal Fragment ◽  
Activity Comparison

SummarySeveral observations suggest that limited proteolysis of full-length 70 kD human thrombopoietin (Tpo) may be important for Tpo biology. Recently, it was reported that thrombin cleaves full-length recombinant human Tpo (rhTpo) sequentially at two sites, Arg195 within the glycan domain followed by Arg117 within the cytokine domain, and that these cleavages modulate Tpo activity in vitro. We demonstrate that urokinase and plasmin also cleave rhTpo in a time-dependent manner. Urokinase cleavage is confined to the glycan domain, and generates a 35 kD N-terminal fragment that contains the intact cytokine domain, and is associated with increased Tpo activity. In contrast, plasmin cleaves Tpo sequentially at two specific sites (Arg205 within the glycan domain followed by Lys52 within the cytokine domain), and is associated with a marked decrease in Tpo activity. These proteolytic events have potential implications for regulation of Tpo activity in vivo.

scholarly journals Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

1999 ◽  
Vol 147 (7) ◽  
pp. 1569-1582 ◽  
Author(s):  
Michelangelo Cordenonsi ◽  
Fabio D'Atri ◽  
Eva Hammar ◽  
David A.D. Parry ◽  
John Kendrick-Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Coiled Coil ◽  
Full Length ◽  
Cytoplasmic Surface ◽  
Terminal Fragment ◽  
Reticulocyte Lysates ◽  
Actomyosin Cytoskeleton

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

The thymic theme of acetylcholinesterase splice variants in myasthenia gravis

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4383-4391 ◽  
Author(s):  
Adi Gilboa-Geffen ◽  
Paul P. Lacoste ◽  
Lilach Soreq ◽  
Geraldine Cizeron-Clairac ◽  
Rozen Le Panse ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Splice Variants ◽  
Thymic Hyperplasia ◽  
Current Therapy ◽  
Cd8 Cells ◽  
Cholinergic Signaling ◽  
Hydrolytic Activities ◽  
Thymic Medulla ◽  
And Function

Abstract Cholinergic signaling and acetylcholinesterase (AChE) influence immune response and inflammation. Autoimmune myasthenia gravis (MG) is mediated by antibodies to the acetylcholine receptor and current therapy is based on anti-AChE drugs. MG is associated with thymic hyperplasia, showing signs of inflammation. The objectives of this study were to analyze the involvement of AChE variants in thymic hyperplasia. We found lower hydrolytic activities in the MG thymus compared with adult controls, accompanied by translocation of AChE-R from the cytoplasm to the membrane and increased expression of the signaling protein kinase PKC-βII. To explore possible causal association of AChE-R changes with thymic composition and function, we used an AChE-R transgenic model and showed smaller thymic medulla compared with strain-matched controls, indicating that AChE-R overexpression interferes with thymic differentiation mechanisms. Interestingly, AChE-R transgenic mice showed increased numbers of CD4+CD8+ cells that were considerably more resistant in vitro to apoptosis than normal thymocytes, suggesting possibly altered positive selection. We further analyzed microarray data of MG thymic hyperplasia compared with healthy controls and found continuous and discrete changes in AChE-annotated GO categories. Together, these findings show that modified AChE gene expression and properties are causally involved in thymic function and development.

scholarly journals Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Bi-Dar Wang ◽  
Kristin Ceniccola ◽  
SuJin Hwang ◽  
Ramez Andrawis ◽  
Anelia Horvath ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
African American ◽  
Rna Splicing ◽  
Splice Variants ◽  
Genome Wide ◽  
Invasive Capacity ◽  
Novel Biomarkers ◽  
Developmental Therapeutics ◽  
Xenograft Models

Abstract Clinical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa.

scholarly journals Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves

1995 ◽  
Vol 311 (3) ◽  
pp. 805-813 ◽  
Author(s):  
C Lenne ◽  
M A Block ◽  
J Garin ◽  
R Douce
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Mass ◽  
Transit Peptide ◽  
Small Heat Shock Protein ◽  
Apparent Molecular Mass ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Small Hsps

A 3 h treatment at 40 degrees C of pea (Pisum sativum var. Douce Provence) plants induces production and accumulation of a small heat-shock protein of 22 kDa apparent molecular mass, designated HSP22, in the matrix compartment of mitochondria [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261]. We show here that the HSP22 precursor (i.e. the mature protein plus the transit peptide) has an apparent molecular mass of 26 kDa after in vitro translation of mRNA extracted from heat-stressed pea plants and immunodetection. We have isolated, cloned and sequenced the full-length cDNA encoding the precursor of the mitochondrial HSP22. An analysis of the amino acid sequence of the mitochondrial HSP22 reveals that this protein is a representative member of the low-molecular-mass heat shock protein (HSP) superfamily, exhibiting the specific consensus regions that are typical of the small HSPs. Most importantly, comparison of the mitochondrial HSP22 sequence with that of chloroplast small HSPs indicates that HSP22 does not contain the typical chloroplast consensus region III. We have also analysed the kinetics of HSP22 induction, and report results on the temporal expression of HSP22 at the transcriptional level. HSP22 mRNA was detected as soon as 10 min after the temperature was raised to a high temperature of 40 degrees C. Then the amount of HSP22 mRNA declined considerably even though pea plants were still submitted to the heat treatment. These results are discussed in light of the translation data previously published [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261], particularly concerning the physiological behaviour of mitochondria when plants are heat-stressed. Furthermore, we have studied the dependence of HSP22 accumulation with temperature and demonstrate that the pea mitochondrial heat-shock response is only developed under extreme environmental growth conditions.

scholarly journals Expression profile and subcellular localization of Torque teno sus virus proteins

2011 ◽  
Vol 92 (10) ◽  
pp. 2446-2457 ◽  
Author(s):  
Laura Martínez-Guinó ◽  
Maria Ballester ◽  
Joaquim Segalés ◽  
Tuija Kekarainen
Keyword(s):  
Subcellular Localization ◽  
Splice Variants ◽  
Full Length ◽  
Protein Isoforms ◽  
Kidney Cells ◽  
Porcine Kidney ◽  
New Information ◽  
Torque Teno Sus Virus

In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV.

scholarly journals Different Forms of DMP1 Play Distinct Roles in Mineralization

2010 ◽  
Vol 89 (4) ◽  
pp. 355-359 ◽  
Author(s):  
A. Gericke ◽  
C. Qin ◽  
Y. Sun ◽  
R. Redfern ◽  
D. Redfern ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Matrix Protein ◽  
Full Length ◽  
Mineralization Process ◽  
Dentin Matrix Protein ◽  
Synthetic Product ◽  
Terminal Fragment ◽  
Mineralized Tissues ◽  
Dentin Matrix

Dentin matrix protein-1 (DMP1) is a major synthetic product of hypertrophic chondrocytes and osteocytes. Previous in vitro studies showed full-length DMP1 inhibits hydroxyapatite (HA) formation and growth, while its N-terminal fragment (37K) promotes HA formation. Since there are 3 fragments within the mineralized tissues [N-terminal, C-terminal (57K), and a chondroitin-sulfate-linked N-terminal fragment (DMP1-PG)], we predicted that each would have a distinct effect on mineralization related to its interaction with HA. In a gelatin-gel system, 37K and 57K fragments were both promoters of HA formation and growth; DMP1-PG was an inhibitor. The secondary structures of the 3 fragments and the full-length protein in the presence and absence of Ca2+ and HA determined by FTIR showed that the full-length protein undergoes slight conformational changes on binding to HA, while 37K, 57K, and DMP1-PG do not change conformation. These findings indicate that distinct forms of DMP1 may work collectively in controlling the mineralization process.

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