A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

Dilated (DCM) and ischemic cardiomyopathies (ICM) are associated with cardiac remodeling, where the ubiquitin–proteasome system (UPS) holds a central role. Little is known about the UPS and its alterations in patients suffering from DCM or ICM. The aim of this study is to characterize the UPS activity in human heart tissue from cardiomyopathy patients. Myocardial tissue from ICM (n = 23), DCM (n = 28), and control (n = 14) patients were used to quantify ubiquitinylated proteins, E3-ubiquitin-ligases muscle-atrophy-F-box (MAFbx)/atrogin-1, muscle-RING-finger-1 (MuRF1), and eukaryotic-translation-initiation-factor-4E (eIF4E), by Western blot. Furthermore, the proteasomal chymotrypsin-like and trypsin-like peptidase activities were determined fluorometrically. Enzyme activity of NAD(P)H oxidase was assessed as an index of reactive oxygen species production. The chymotrypsin- (p = 0.71) and caspase-like proteasomal activity (p = 0.93) was similar between the groups. Trypsin-like proteasomal activity was lower in ICM (0.78 ± 0.11 µU/mg) compared to DCM (1.06 ± 0.08 µU/mg) and control (1.00 ± 0.06 µU/mg; p = 0.06) samples. Decreased ubiquitin expression in both cardiomyopathy groups (ICM vs. control: p < 0.001; DCM vs. control: p < 0.001), as well as less ubiquitin-positive deposits in ICM-damaged tissue (ICM: 4.19% ± 0.60%, control: 6.28% ± 0.40%, p = 0.022), were detected. E3-ligase MuRF1 protein expression (p = 0.62), NADPH-oxidase activity (p = 0.63), and AIF-positive cells (p = 0.50). Statistical trends were detected for reduced MAFbx protein expression in the DCM-group (p = 0.07). Different levels of UPS components, E3 ligases, and UPS activation markers were observed in myocardial tissue from patients affected by DCM and ICM, suggesting differential involvement of the UPS in the underlying pathologies.

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Altered expression of selected genes in kidney of rats with lithium-induced NDI

AJP Renal Physiology ◽

10.1152/ajprenal.00305.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. F1276-F1289 ◽

Cited By ~ 45

Author(s):

Aleksandra Rojek ◽

Jakob Nielsen ◽

Heddwen L. Brooks ◽

Hong Gong ◽

Young-Hee Kim ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Cellular Proliferation ◽

Collecting Duct ◽

Lithium Treatment ◽

Altered Expression ◽

Ezrin Expression ◽

Microarray Screening ◽

And Control ◽

Thin Limb

Lithium treatment is associated with development of nephrogenic diabetes insipidus, caused in part by downregulation of collecting duct aquaporin-2 (AQP2) and AQP3 expression. In the present study, we carried out cDNA microarray screening of gene expression in the inner medulla (IM) of lithium-treated and control rats, and selected genes were then investigated at the protein level by immunoblotting and/or immunohistochemistry. The following genes exhibited significantly altered transcription and mRNA expression levels, and these were compatible with the changes in protein expression. 11β-Hydroxysteroid dehydrogenase type 2 protein expression in the IM was markedly increased (198 ± 25% of controls, n = 6), and immunocytochemistry demonstrated an increased labeling of IM collecting duct (IMCD) principal cells. This indicated altered renal mineralocorticoid/glucocorticoid responses in lithium-treated rats. The inhibitor of cyclin-dependent kinases p27 (KIP) protein expression was significantly decreased or undetectable in the IMCD cells, pointing to increased cellular proliferation and remodeling. Heat shock protein 27 protein expression was decreased in the IM (64 ± 6% of controls, n = 6), likely to be associated with the decreased medullary osmolality in lithium-treated rats. Consistent with this, lens aldose reductase protein expression was markedly decreased in the IM (16 ± 2% of controls, n = 6), and immunocytochemistry revealed decreased expression in the thin limb cells in the middle and terminal parts of the IM. Ezrin protein expression was upregulated in the IM (158 ± 16% of controls, n = 6), where it was predominantly expressed in the apical and cytoplasmic domain of the IMCD cells. Increased ezrin expression indicated remodeling of the actin cytoskeleton and/or altered regulation of IMCD transporters. In conclusion, the present study demonstrates changes in gene expression not only in the collecting duct but also in the thin limb of the loop of Henle in the IM, and several of these genes are linked to altered sodium and water reabsorption, cell cycling, and changes in interstitial osmolality.

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Transcriptome Profiling Reveals New Insights into the Immune Microenvironment and Upregulation of Novel Biomarkers in Metastatic Uveal Melanoma

Cancers ◽

10.3390/cancers12102832 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2832 ◽

Cited By ~ 2

Author(s):

Yamini Krishna ◽

Amelia Acha-Sagredo ◽

Dorota Sabat-Pośpiech ◽

Natalie Kipling ◽

Kim Clarke ◽

...

Keyword(s):

Protein Expression ◽

Uveal Melanoma ◽

Transcriptome Profiling ◽

Epithelioid Cell ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Galectin 3 ◽

Novel Biomarkers ◽

And Control ◽

Infiltrating Lymphocytes

Metastatic uveal melanoma (mUM) to the liver is incurable. Transcriptome profiling of 40 formalin-fixed paraffin-embedded mUM liver resections and 6 control liver specimens was undertaken. mUMs were assessed for morphology, nuclear BAP1 (nBAP1) expression, and their tumour microenvironments (TME) using an “immunoscore” (absent/altered/high) for tumour-infiltrating lymphocytes (TILs) and macrophages (TAMs). Transcriptomes were compared between mUM and control liver; intersegmental and intratumoural analyses were also undertaken. Most mUM were epithelioid cell-type (75%), amelanotic (55%), and nBAP1-ve (70%). They had intermediate (68%) or absent (15%) immunoscores for TILs and intermediate (53%) or high (45%) immunoscores for TAMs. M2-TAMs were dominant in the mUM-TME, with upregulated expression of ANXA1, CD74, CXCR4, MIF, STAT3, PLA2G6, and TGFB1. Compared to control liver, mUM showed significant (p < 0.01) upregulation of 10 genes: DUSP4, PRAME, CD44, IRF4/MUM1, BCL2, CD146/MCAM/MUC18, IGF1R, PNMA1, MFGE8/lactadherin, and LGALS3/Galectin-3. Protein expression of DUSP4, CD44, IRF4, BCL-2, CD146, and IGF1R was validated in all mUMs, whereas protein expression of PRAME was validated in 10% cases; LGALS3 stained TAMs, and MFGEF8 highlighted bile ducts only. Intersegmental mUMs show differing transcriptomes, whereas those within a single mUM were similar. Our results show that M2-TAMs dominate mUM-TME with upregulation of genes contributing to immunosuppression. mUM significantly overexpress genes with targetable signalling pathways, and yet these may differ between intersegmental lesions.

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Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.

The Journal of Cell Biology ◽

10.1083/jcb.99.6.2146 ◽

1984 ◽

Vol 99 (6) ◽

pp. 2146-2156 ◽

Cited By ~ 34

Author(s):

R J Leslie ◽

W M Saxton ◽

T J Mitchison ◽

B Neighbors ◽

E D Salmon ◽

...

Keyword(s):

Living Cells ◽

Before And After ◽

Ion Laser ◽

Cross Links ◽

Protein Incorporation ◽

And Control ◽

Argon Ion ◽

Brain Tubulin ◽

Control Protein

Brain tubulin has been conjugated with dichlorotriazinyl-aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, however, DTAF-microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.

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Reduced axonal motor protein expression in non-lesional grey matter in multiple sclerosis

Multiple Sclerosis Journal ◽

10.1177/1352458513508836 ◽

2013 ◽

Vol 20 (7) ◽

pp. 812-821 ◽

Cited By ~ 14

Author(s):

K Hares ◽

K Kemp ◽

C Rice ◽

E Gray ◽

N Scolding ◽

...

Keyword(s):

Multiple Sclerosis ◽

Protein Expression ◽

Axonal Transport ◽

Neurological Disease ◽

Grey Matter ◽

Messenger Ribonucleic Acid ◽

Protein Levels ◽

Anterograde Axonal Transport ◽

Kinesin Superfamily ◽

And Control

Background: Multiple sclerosis (MS) is a neurological disease characterised by central nervous system inflammation, demyelination, axonal degeneration and neuronal injury. Preventing neuronal and axon damage is of paramount importance in attempts to prevent disease progression. Intact axonal transport mechanisms are crucial to axonal integrity and evidence suggests these mechanisms are disrupted in MS. Anterograde axonal transport is mediated to a large extent through the kinesin superfamily proteins. Recently, certain kinesin superfamily proteins (KIF5A, KIF1B and KIF21B) were implicated in MS pathology. Objectives: To investigate the expression of KIF5A, KIF21B and KIF1B in MS and control post-mortem grey matter. Methods: Using both quantitative real-time polymerase chain reaction (PCR) and Immunodot-blots assays, we analysed the expression of kinesin superfamily proteins in 27 MS cases and 13 control cases not linked to neurological disease. Results: We have shown significant reductions in KIF5A, KIF21B and KIF1B messenger ribonucleic acid (mRNA) expression and also KIF5A protein expression in MS grey matter, as compared to control grey matter. Conclusion: We have shown significant reductions in mRNA and protein levels of axonal motor proteins in the grey matter of MS cases, which may have important implications for the pathogenesis of neuronal/axonal injury in the disease.

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Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook ofSalmonella

Journal of Bacteriology ◽

10.1128/jb.181.18.5808-5813.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5808-5813 ◽

Cited By ~ 32

Author(s):

Kazumasa Muramoto ◽

Shigeru Makishima ◽

Shin-Ichi Aizawa ◽

Robert M. Macnab

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Basal Bodies ◽

Wild Type ◽

Hook Length ◽

Capping Protein ◽

Protein Levels ◽

Junction Protein ◽

And Control ◽

Control Protein

ABSTRACT The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, afliC (flagellin) mutant, or a fliD(hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

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SUZ12 Participates in PNH Cloning Proliferation By Regulating Histone H3K27me3 Methylation Level

Blood ◽

10.1182/blood-2021-148546 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1106-1106

Author(s):

Rong Fu ◽

Yingying Chen ◽

Zonghong Shao ◽

Hui Liu ◽

Lijie Zeng ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Protein Expression ◽

Peripheral Blood ◽

Methylation Level ◽

Cell Model ◽

Gene Mutations ◽

Control Group ◽

Mrna And Protein Expression ◽

And Control

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a disease of hematopoietic stem cell membrane defects due to acquired PIG-Amutation. Our previous study found some secondary gene mutations in PNH patients by WES. However, it is not clear exactly which mutations are associated with the disease. So, 97 target genes were selected as a target gene panel and tested in 23 PNH patients by DNA sequencing of specific target regions. We found that all PNH patients had other gene mutations except PIG-Amutations, including TTN, NCOR2, CPS1, MUC4, SUZ12, LFNG, CELSR2, JAK2, SETBP1 and KMT2D (Figure1A). Through harmful analysis, KEGG enrichment, GO enrichment analysis and protein interaction analysis, we screened out the secondary mutant gene SUZ12 that may be involved in the cloning proliferation of PNH. We detected the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in PNH patients and health volunteers, the results showed that the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in peripheral blood CD59 -cells of PNH patients were higher than those in CD59 + cells of PNH patients and healthy controls (Figure1B). The relative expression level of SUZ12 in peripheral blood CD59 -cells of PNH patients was correlated with (r=0.4162, p=0.0385), CD59 -erythrocyte ratio (r=0.4636, p=0.0196), CD59 -monocyte ratio (r=0.4052, p=0.0495), Flaer -monocyte ratio (r=0.6769, p=0.0004) and Flaer -granulocytic ratio (r=0.6146, p=0.0018), indicating that SUZ12 may be involved in abnormal PNH cloning and proliferation by regulating H3K27me3. To verify the role of SUZ12 in the proliferation of PNH cloning, we used CRISPR/Cas9 to knockdown PIG-A expression in THP-1 cells to construct A PNH cell model, the expression level of PIG-A protein in the cell model was significantly decreased, and the proportion of CD59 - cells accounted was stable at 95%. Then lentivirus transfection was used to knockdown the expression of SUZ12 in PNH cell model. The results showed when the SUZ12 expression was knockdown, the methylation level of histone H3K27me3 was decreased, the cell proliferation activity was decreased, apoptosis was increased, and the cell cycle was arrested at G0/G1 phase. The proportion of CD59 + cells increased gradually from 3 weeks after transfection, and significantly increased at 4 weeks after transfection, while no changes were observed in the empty virus group and control group (Figure1C). Four weeks after lentivirus transfection, the expression of PIG-A protein recovered in SUZ12 knockdown group compared with empty virus group and control group (Figure1D). In conclusion, SUZ12 mutation leads to the overexpression of SUZ12, which can affect cell proliferation, apoptosis and cell cycle by regulating the methylation level of histone H3K27me3, thereby promoting the proliferation of PNH abnormal cloning and participating in the pathogenesis of PNH. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Polyomic Analyses of Dopaminergic Neurons Isolated From Human Substantia Nigra in Parkinson’s Disease: An Exploratory Study.

10.21203/rs.3.rs-182873/v1 ◽

2021 ◽

Author(s):

Affif ZACCARIA ◽

Paola Antinori Malaspina ◽

Virginie Licker ◽

Enikö Kovari ◽

Johannes A Lobrinus ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Substantia Nigra ◽

Protein Expression ◽

Differential Expression ◽

Exploratory Study ◽

Substantia Nigra Pars Compacta ◽

Control Group ◽

Gene And Protein Expression ◽

And Control

Abstract Background Dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) selectively and progressively degenerate in Parkinson’s disease (PD). Until now, molecular analyses of DA neurons in PD have been limited to genomic and transcriptomic approaches, whereas, to the best of our knowledge, no proteomic or combined polyomic study examining the protein profile of these neurons, is currently available. Methods In this exploratory study, we used laser microdissection to extract DA neurons from 10 human SNpc samples obtained at autopsy in PD patients and control subjects. Extracted RNA and proteins were identified by RNA sequencing and nano-LC-MS/MS, respectively, and the differential expression between the PD and control group was assessed. Results Qualitative analyses confirmed that the microdissection protocol preserves the integrity of our samples and offers access to specific molecular pathways. This polyomic analysis highlighted differential expression of 52 genes and 33 proteins, including molecules of interest already known to be dysregulated in PD, such as LRP2, PNMT, CXCR4, MAOA and CBLN1 genes, or the Aldehyde dehydrogenase 1 protein. On the other hand, despite the same samples were used for both analyses, correlation between RNA and protein expression was low, as exemplified by the CST3 gene encoding for the cystatin C protein. Conclusion This is the first exploratory study analyzing both gene and protein expression of LMD-dissected DA neurons from SNpc in PD. Although correlation between RNA and protein expressions was limited, this polyomic study provides an extensive and integrated overview of molecular changes identified in the PD SNpc and may offer novel insights into specific pathological processes at work in PD degeneration.

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A synaptic threshold mechanism for computing escape decisions

10.1101/272492 ◽

2018 ◽

Cited By ~ 1

Author(s):

D.A Evans ◽

A.V. Stempel ◽

R. Vale ◽

S. Ruehle ◽

Y. Lefler ◽

...

Keyword(s):

Network Activity ◽

Biophysical Model ◽

Sensory Stimuli ◽

Recurrent Excitation ◽

Threshold Mechanism ◽

Threat Level ◽

Deep Layers ◽

Defensive Behaviours ◽

And Control ◽

The Brain

Escaping from imminent danger is an instinctive behaviour fundamental for survival that requires classifying sensory stimuli as harmless or threatening. The absence of threat allows animals to forage for essential resources, but as the level of threat and potential for harm increases, they have to decide whether or not to seek safety1. Despite previous work on instinctive defensive behaviours in rodents2–13, little is known about how the brain computes the threat level for initiating escape. Here we show that the probability and vigour of escape in mice scale with the intensity of innate threats, and are well described by a theoretical model that computes the distance between threat level and an escape threshold. Calcium imaging and optogenetics in the midbrain of freely behaving mice show that the activity of excitatory VGluT2+ neurons in the deep layers of the medial superior colliculus (mSC) represents the threat stimulus intensity and is predictive of escape, whereas dorsal periaqueductal gray (dPAG) VGluT2+ neurons encode exclusively the escape choice and control escape vigour. We demonstrate a feed-forward monosynaptic excitatory connection from mSC to dPAG neurons that is weak and unreliable, yet necessary for escape behaviour, and which we suggest provides a synaptic threshold for dPAG activation and the initiation of escape. This threshold can be overcome by high mSC network activity because of short-term synaptic facilitation and recurrent excitation within the mSC, which amplifies and sustains synaptic drive to the dPAG. Thus, dPAG VGluT2+ neurons compute escape decisions and vigour using a synaptic mechanism to threshold threat information received from the mSC, and provide a biophysical model of how the brain performs a critical behavioural computation.

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