scholarly journals Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Meng-ya Geng ◽  
Lizhuo Wang ◽  
Ying-ying Song ◽  
Jing Gu ◽  
Xin Hu ◽  
...  
Keyword(s):  
Degradation Pathway ◽  
Normal Function ◽  
Mrna Levels ◽  
Kidney Structure ◽  
Protein Levels ◽  
Basement Membrane Thickening ◽  
Foot Process ◽  
Autophagy Pathway ◽  
Lysosomal Membrane Protein ◽  
And Function

AbstractThe regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2−/− mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, and lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2−/− cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.

scholarly journals Restoration of Noradrenergic Function in Parkinson’s Disease Model Mice

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110097
Author(s):  
Kui Cui ◽  
Fan Yang ◽  
Turan Tufan ◽  
Muhammad U. Raza ◽  
Yanqiang Zhan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Transcription Factors ◽  
Disease Model ◽  
Neuronal Loss ◽  
Mrna Levels ◽  
Gene Therapies ◽  
Protein Levels ◽  
Dopaminergic Systems ◽  
And Function

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson’s disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia

Brain ◽  
2019 ◽  
Vol 142 (11) ◽  
pp. 3382-3397 ◽  
Author(s):  
Frédéric M Vaz ◽  
John H McDermott ◽  
Mariëlle Alders ◽  
Saskia B Wortmann ◽  
Stefan Kölker ◽  
...  
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Membrane Lipids ◽  
Cerebellar Atrophy ◽  
Global Developmental Delay ◽  
Mrna Levels ◽  
Spastic Paraplegia ◽  
Protein Levels ◽  
And Function ◽  
Lipid Abnormalities ◽  
The Brain

Abstract CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

scholarly journals TcpH Influences Virulence Gene Expression in Vibrio cholerae by Inhibiting Degradation of the Transcription Activator TcpP

2004 ◽  
Vol 186 (24) ◽  
pp. 8309-8316 ◽  
Author(s):  
Nancy A. Beck ◽  
Eric S. Krukonis ◽  
Victor J. DiRita
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Time Course ◽  
Virulence Gene ◽  
Degradation Pathway ◽  
Mrna Levels ◽  
Transcription Activator ◽  
Virulence Gene Expression ◽  
Protein Levels ◽  
Periplasmic Domain

ABSTRACT Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.

scholarly journals Alcohol Metabolism in the Progression of Human Nonalcoholic Steatohepatitis

2018 ◽  
Vol 164 (2) ◽  
pp. 428-438 ◽  
Author(s):  
Hui Li ◽  
Erica Toth ◽  
Nathan J Cherrington
Keyword(s):  
Nonalcoholic Steatohepatitis ◽  
Protein Level ◽  
Mrna Levels ◽  
Alcohol Metabolism ◽  
Aldh Activity ◽  
Nonalcoholic Fatty Liver ◽  
Protein Levels ◽  
Protein Adduct ◽  
And Function ◽  
Adh Activity

Abstract Alcohol metabolism is a well-characterized biological process that is dominated by the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) families. Nonalcoholic steatohepatitis (NASH) is the advanced inflammatory stage of nonalcoholic fatty liver disease (NAFLD) and is known to alter the metabolism and disposition of numerous drugs. The purpose of this study was to investigate the alterations in alcohol metabolism processes in response to human NASH progression. Expression and function of ADHs, ALDHs, and catalase were examined in normal, steatosis, NASH (fatty) and NASH (not fatty) human liver samples. ALDH4A1 mRNA was significantly decreased in both NASH groups, while no significant changes were observed in the mRNA levels of other alcohol-related enzymes. The protein levels of ADH1A, ADH1B, and ADH4 were each decreased in the NASH groups, which was consistent with a decreased overall ADH activity. The protein level of ALDH2 was significantly increased in both NASH groups, while ALDH1A1 and ALDH1B1 were only decreased in NASH (fatty) samples. ALDH activity represented by oxidation of acetaldehyde was decreased in the NASH (fatty) group. The protein level of catalase was decreased in both NASH groups, though activity was unchanged. Furthermore, the significant accumulation of 4-hydroxynonenal protein adduct in NASH indicated significant oxidative stress and a potential reduction in ALDH activity. Collectively, ADH and ALDH expression and function are profoundly altered in the progression of NASH, which may have a notable impact on ADH- and ALDH-associated cellular metabolism processes and lead to significant alterations in drug metabolism mediated by these enzymes.

scholarly journals Synaptic activity controls autophagic vacuole motility and function in dendrites

2021 ◽  
Vol 220 (6) ◽  
Author(s):  
Vineet Vinay Kulkarni ◽  
Anip Anand ◽  
Jessica Brandt Herr ◽  
Christina Miranda ◽  
Maria Chalokh Vogel ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Degradation Pathway ◽  
Autophagic Vacuole ◽  
Synaptic Activity ◽  
Lysosomal Degradation ◽  
Autophagy Pathway ◽  
Dynamics And Function ◽  
And Function ◽  
Activity Dependent ◽  
Stimulation Of

Macroautophagy (hereafter “autophagy”) is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.

scholarly journals VDR/Atg3 Axis Regulates Slit Diaphragm to Tight Junction Transition via p62-mediated Autophagy Pathway in Diabetic Nephropathy

2021 ◽  
Author(s):  
Bin Wang ◽  
Jing-yi Qian ◽  
Tao-tao Tang ◽  
Li-lu Lin ◽  
Nan Yu ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Tight Junction ◽  
Diabetic Animal ◽  
Slit Diaphragm ◽  
Autophagic Flux ◽  
The Novel ◽  
Foot Process ◽  
Autophagy Pathway ◽  
And Function ◽  
Early Diabetic Nephropathy

Foot process effacement is an important feature of early diabetic nephropathy (DN) which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62 mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase 3 and caspase 8. Interestingly, the expression of VDR in podocytes was decreased under diabetic condition which impaired autophagic flux through down-regulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking p62-mediated autophagy pathway in DN.<br>

Differential regulation of G protein expression in rat hearts exposed to chronic hypoxia

1995 ◽  
Vol 269 (6) ◽  
pp. H1865-H1873 ◽  
Author(s):  
R. Kacimi ◽  
J. M. Moalic ◽  
A. Aldashev ◽  
D. E. Vatner ◽  
J. P. Richalet ◽  
...  
Keyword(s):  
Gene Expression ◽  
G Protein ◽  
Functional Activity ◽  
Protein Level ◽  
Chronic Hypoxia ◽  
Mrna Level ◽  
Mrna Levels ◽  
Protein Levels ◽  
Rat Hearts ◽  
And Function

Chronic hypoxia impairs adrenergic responsiveness. A modulation of Gs and/or G1 protein alpha-subunits may be associated with the downregulation of the beta-adrenergic receptors previously found in chronic hypoxia. G protein gene expression and protein level and function in rat hearts exposed to a 30-day hypobaric chronic hypoxia were compared with control rat hearts. No change was observed in G alpha s mRNA levels in either right or left ventricles. In right ventricles, mRNA levels of G alpha i-2 increased by 40% (P < 0.05), but not in left ventricles. In both left and right ventricles, chronic hypoxia did not modify G alpha i-2 and G alpha s protein amounts, but significantly decreased functional activity of G alpha s. In conclusion, gene expression, protein levels of G alpha s and G alpha i-2, and activity of G alpha s do not change in parallel fashion with chronic hypoxia. In chronic hypoxic right ventricles, although the mRNA level of G alpha i-2 is increased, the protein level is unchanged. One potential mechanism of desensitization to catecholamines in chronic hypoxia appears to involve a decreased functional activity of G alpha s in spite of normal mRNA and protein levels

scholarly journals O-Polysaccharide Glycosylation Is Required for Stability and Function of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans

2012 ◽  
Vol 80 (8) ◽  
pp. 2868-2877 ◽  
Author(s):  
Gaoyan Tang ◽  
Teresa Ruiz ◽  
Keith P. Mintz
Keyword(s):  
Cellular Localization ◽  
Proteolytic Enzymes ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Binding Activity ◽  
Mrna Levels ◽  
Collagen Binding ◽  
Content Type ◽  
Protein Levels ◽  
Mutant Strains ◽  
And Function

ABSTRACTAggregatibacter actinomycetemcomitansis hypothesized to colonize through the interaction with collagen and establish a reservoir for further dissemination. The trimeric adhesin EmaA ofA. actinomycetemcomitansbinds to collagen and is modified with sugars mediated by an O-antigen polysaccharide ligase (WaaL) that is associated with lipopolysaccharide (LPS) biosynthesis (G. Tang and K. Mintz, J. Bacteriol.192:1395–1404, 2010). This investigation characterized the function and cellular localization of EmaA glycosylation. The interruption of LPS biogenesis by using genetic and pharmacological methods changed the amount and biophysical properties of EmaA molecules in the outer membrane. InrmlCandwaaLmutant strains, the membrane-associated EmaA was reduced by 50% compared with the wild-type strain, without changes in mRNA levels. The membrane-associated EmaA protein levels were recovered by complementation with the corresponding O-polysaccharide (O-PS) biosynthetic genes. In contrast, another trimeric autotransporter, epithelial adhesin ApiA, was not affected in the same mutant background. The inhibition of undecaprenyl pyrophosphate recycling by bacitracin resulted in a similar decrease in the membrane-associated EmaA protein. This effect was reversed by removal of the compound. A significant decrease in collagen binding activity was observed in strains expressing the nonglycosylated form of EmaA. Furthermore, the electrophoretic mobility shifts of the EmaA monomers found in the O-PS mutant strains were associated only with the membrane-associated protein and not with the cytoplasmic pre-EmaA protein, suggesting that this modification does not occur in the cytoplasm. The glycan modification of EmaA appears to be required for collagen binding activity and protection of the protein against degradation by proteolytic enzymes.

scholarly journals Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1603-1606 ◽  
Author(s):  
Elena E. Solomou ◽  
Katayoun Rezvani ◽  
Stephan Mielke ◽  
Daniela Malide ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
T Regulatory Cells ◽  
T Cell Development ◽  
Foxp3 Expression ◽  
Mrna Levels ◽  
Regulatory Cells ◽  
Protein Levels ◽  
Almost All ◽  
And Function

Abstract Regulatory T cells are believed to control the development and progression of autoimmunity by suppressing autoreactive T cells. Decreased numbers of CD4+CD25+ FOXP3+ T cells (Tregs) are associated with impaired immune homeostasis and development of autoimmune diseases. The transcription factors FOXP3 and NFAT1 have key roles in regulatory T-cell development and function. We show that Tregs are decreased at presentation in almost all patients with aplastic anemia; FOXP3 protein and mRNA levels also are significantly lower in patients with aplastic anemia and NFAT1 protein levels are decreased or absent. Transfection of FOXP3-deficient CD4+CD25+ T cells from patients with a plasmid encoding wild-type NFAT1 resulted in increased FOXP3 expression in these cells. By NFAT1 knockdown in CD4+CD25+ T cells, FOXP3 expression was decreased when NFAT1 expression was decreased. Our findings indicate that decreased NFAT1 could explain low FOXP3 expression and diminished Treg frequency in aplastic anemia. Treg defects are now implicated in autoimmune marrow failure.

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