scholarly journals Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases inSchistosoma mansoni

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Roberta Verciano Pereira ◽  
Fernanda Janku Cabral ◽  
Matheus de Souza Gomes ◽  
Liana Konovaloff Jannotti-Passos ◽  
William Castro-Borges ◽  
...  
Keyword(s):  
Protein Localization ◽  
Cell Cycle Control ◽  
Developmentally Regulated ◽  
Cellular Processes ◽  
Significant Difference ◽  
Endopeptidase Activity ◽  
Regulated Expression ◽  
Differential Gene

Small ubiquitin-related modifier (SUMO) is involved in numerous cellular processes including protein localization, transcription, and cell cycle control. SUMOylation is a dynamic process, catalyzed by three SUMO-specific enzymes and reversed by Sentrin/SUMO-specific proteases (SENPs). Here we report the characterization of these proteases inSchistosoma mansoni. Usingin silicoanalysis, we identified two SENPs sequences, orthologs of mammalian SENP1 and SENP7, confirming their identities and conservation through phylogenetic analysis. In addition, the transcript levels ofSmsenp1/7in cercariae, adult worms, andin vitrocultivated schistosomula were measured by qRT-PCR. Our data revealed upregulation of theSmsenp1/7transcripts in cercariae and early schistosomula, followed by a marked differential gene expression in the other analyzed stages. However, no significant difference in expression profile between the paralogs was observed for the analyzed stages. Furthermore, in order to detect deSUMOylating capabilities in crude parasite extracts,SmSENP1 enzymatic activity was evaluated using SUMO-1-AMC substrate. The endopeptidase activity related to SUMO-1 precursor processing did not differ significantly between cercariae and adult worms. Taken together, these results support the developmentally regulated expression of SUMO-specific proteases inS. mansoni.

scholarly journals Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

2005 ◽  
Vol 16 (5) ◽  
pp. 2129-2138 ◽  
Author(s):  
Frederick R. Cross ◽  
Lea Schroeder ◽  
Martin Kruse ◽  
Katherine C. Chen
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Predictive Ability ◽  
Eukaryotic Cell ◽  
Model Predictions ◽  
Mitotic Cyclin ◽  
Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

scholarly journals The Developmentally Regulated Expression of Twisted Gastrulation Reveals a Role for Bone Morphogenetic Proteins in the Control of T Cell Development

2002 ◽  
Vol 196 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Daniel Graf ◽  
Suran Nethisinghe ◽  
Donald B. Palmer ◽  
Amanda G. Fisher ◽  
Matthias Merkenschlager
Keyword(s):  
T Cell ◽  
Bone Morphogenetic Proteins ◽  
Cell Receptor ◽  
Mammalian Development ◽  
Double Positive ◽  
Developmentally Regulated ◽  
Twisted Gastrulation ◽  
Thymocyte Proliferation ◽  
Regulated Expression

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Identification and characterization of novel filament-forming proteins in cyanobacteria

2019 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Christian Woehle ◽  
Julia Weissenbach ◽  
Andreas O. Helbig ◽  
Tal Dagan ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Cytoskeletal Proteins ◽  
Tetratricopeptide Repeat ◽  
Cellular Processes ◽  
Tetratricopeptide Repeat Protein ◽  
Pcc 7120 ◽  
Pcc 7942

AbstractFilament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains

2020 ◽  
Vol 21 (15) ◽  
pp. 5351
Author(s):  
Methanee Hiranyakorn ◽  
Saeko Yanaka ◽  
Tadashi Satoh ◽  
Thunchanok Wilasri ◽  
Benchawan Jityuti ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Hydrophobic Surface ◽  
Enzymatic Reactions ◽  
Multidomain Proteins ◽  
Solvent Exposure ◽  
Hydrophobic Surfaces ◽  
Cellular Processes ◽  
Nmr Characterization

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

scholarly journals Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anabel Zabala-Peñafiel ◽  
Geovane Dias-Lopes ◽  
Léa Cysne-Finkelstein ◽  
Fátima Conceição-Silva ◽  
Luciana de Freitas Campos Miranda ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Principal Component ◽  
Clinical Isolates ◽  
In Vitro Susceptibility ◽  
Axenic Amastigotes ◽  
Homogeneous Cluster ◽  
Significant Difference ◽  
American Tegumentary Leishmaniasis

AbstractGlucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

180 EXPRESSION OF TUMOR NECROSIS FACTOR-STIMULATED GENE 6 PROTEIN IN BOVINE CUMULUS–OOCYTE COMPLEXES AT DIFFERENT MATURATION STATUS

2013 ◽  
Vol 25 (1) ◽  
pp. 239
Author(s):  
P. Trzeciak ◽  
R. Starzyński ◽  
Ł. Rąpała ◽  
S. Dąbrowski ◽  
E. Nałęcz-Nieniewska ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Protein Localization ◽  
Statistical Significance ◽  
Mrna Level ◽  
Fluorescent Microscopy ◽  
Cumulus Cells ◽  
Mean Values ◽  
Significant Difference ◽  
Tukey Honestly Significant Difference

The TSG-6 is a ~35-kDa protein belonging to hyaluronan binding superfamily proteins. The TSG-6 plays role in inflammation and in inflammation-like processes (i.e. ovulation). The tsg-6 expression is induced in cumulus–oocyte complex (COC) cells just before ovulation. It is involved in the migration of cumulus cells though the formation and stabilisation of the extracellular matrix. Disturbances in secretion of this protein lead to a reduction in the number of ovulated oocytes. Although studies of the prevalence and role of TSG-6 in COC were conducted in several animal models, little is known about TSG-6 in cattle. The aim of this study was to assess tsg-6 mRNA expression and protein localization in bovine cumulus cells from COC at different maturation status. Ovaries were collected from the slaughterhouse. Cumulus cells were isolated from COCs in different stages of maturity. In variant I, COC were isolated from small follicles of Φ 2 to 6 mm. In variant II, COC were also isolated from small follicles and matured in vitro in TCM199 HEPES with 10% FBS and 0.02 IU NIH-pFSH mL–1, 1 µg mL–1 β-oestradiol, 0.2 µM sodium pyruvate, 50 µg mL–1 gentamicin in 5% CO2, 38.5°C for 24 h. In variant III, COCs were isolated from large follicles of Φ >15 mm. Analysis of tsg-6 expression in cumulus cells was performed using real-time PCR. Expression of Tsg-6 was normalized to that of s18. Statistical analysis of tsg-6 mRNA level in all variants was carried out by 1-way ANOVA, and comparisons of mean values were made with the Tukey honestly significant difference test (Statgraphic 5.1 Centurion); P < 0.05 was considered to reflect the presence of statistical significance. For all variants, paraffin-embedded slices of COC were carried out to localise TSG-6 by indirect method of immunofluorescence. Immunostaining of the TSG-6 protein was performed using primary polyclonal antibody raised against bovine TSG-6. Antigen–antibody complexes were visualised after incubation with secondary IgG conjugated with fluorescein isothiocynanate. Immunolocalization of TSG-6 was performed using fluorescent microscopy. The relative expression of tsg-6 mRNA in variant II was more than 2 times higher (1.887 ± 0.797 a.u.) compared with variant III (0.760 ± 0.130 a.u.). The difference was statistically significant (P < 0.05). No tsg-6 expression in variant I was detected. The presence of TSG-6 protein in the extracellular matrix of cumulus cells from variant II as well as from variant III was detected. The present data suggest that tsg-6 gene expression and its protein presence are stimulated during COC maturation in cattle as previously demonstrated for other species. Supported by Warsaw University of Life Sciences, Faculty of Veterinary Medicine: 505-10-02330050.

Developmentally regulated activation of apoptosis early in Xenopus gastrulation results in cyclin A degradation during interphase of the cell cycle

Development ◽  
1997 ◽  
Vol 124 (16) ◽  
pp. 3185-3195 ◽  
Author(s):  
J.H. Stack ◽  
J.W. Newport
Keyword(s):  
Cyclin A ◽  
Developmentally Regulated ◽  
Apoptotic Pathway ◽  
Cellular Processes ◽  
Xenopus Embryos ◽  
Zygotic Transcription ◽  
Cellular Apoptosis ◽  
The Stability ◽  
Critical Developmental Period

Previous work identified a developmental timer that controls the stability of cyclin A protein in interphase-arrested Xenopus embryos. It was shown that cyclins A1 and A2 abruptly become unstable in hydroxyurea-treated embryos at the time that untreated embryos are beginning gastrulation (early gastrulation transition; EGT). We have demonstrated here that cyclins A1 and A2 are degraded at the equivalent of the EGT by the ICE-like caspases that are responsible for programmed cell death or apoptosis. Analysis of embryos treated with hydroxyurea or cycloheximide showed widespread cellular apoptosis coincident with cyclin A cleavage. Our data further indicate that the apoptotic pathway is present in Xenopus embryos prior to the EGT; however, it is maintained in an inactive state in early cleaving embryos by maternally encoded inhibitors. Characterization of the timing of the activation of apoptosis implicates the initiation of zygotic transcription at the mid-blastula transition (MBT) in the suppression of apoptosis in normal embryos. The decreased biosynthetic capacity of embryos treated with hydroxyurea or cycloheximide most likely interferes with the ability to maintain sufficient levels of apoptotic inhibitors and results in widespread apoptosis. Our results suggest a scenario whereby the apoptotic pathway is suppressed in the early cleaving embryo by maternally contributed inhibitors. Degradation at the EGT of maternal RNAs encoding these inhibitors is compensated for by new zygotic transcription beginning at the MBT. This indicates that the interval between the MBT and the EGT represents a critical developmental period during which the regulation of embryonic cellular processes is transferred from maternal to zygotic control.

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