HSPB5 engages multiple states of a destabilized client to enhance chaperone activity in a stress-dependent manner

AbstractMissense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson’s disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal–lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

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Oxidative stress increases megalin expression in the renal proximal tubules during the normoalbuminuric stage of diabetes mellitus

AJP Renal Physiology ◽

10.1152/ajprenal.00108.2017 ◽

2018 ◽

Vol 314 (3) ◽

pp. F462-F470 ◽

Cited By ~ 5

Author(s):

Yoshifumi Kurosaki ◽

Akemi Imoto ◽

Fumitaka Kawakami ◽

Masanori Yokoba ◽

Tsuneo Takenaka ◽

...

Keyword(s):

Oxidative Stress ◽

Diabetes Mellitus ◽

Hydrogen Peroxide ◽

High Glucose ◽

Renal Cortex ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Phosphorylated Akt ◽

Stress Dependent ◽

Phosphatidylinositol 3

Megalin, an endocytic receptor expressed in proximal tubule cells, plays a critical role in renal tubular protein reabsorption and is associated with the albuminuria observed in diabetic nephropathy. We have previously reported increased oxidant production in the renal cortex during the normoalbuminuric stage of diabetes mellitus (DM); however, the relationship between oxidative stress and renal megalin expression during the normoalbuminuric stage of DM remains unclear. In the present study, we evaluated whether oxidative stress affects megalin expression in the normoalbuminuric stage of DM in a streptozotocin-induced diabetic rat model and in immortalized human proximal tubular cells (HK-2). We demonstrated that increased expression of renal megalin accompanies oxidative stress during the early stage of DM, before albuminuria development. Telmisartan treatment prevented the diabetes-induced elevation in megalin level, possibly through an oxidative stress-dependent mechanism. In HK-2 cells, hydrogen peroxide significantly increased megalin levels in a dose- and time-dependent manner; however, the elevation in megalin expression was decreased following prolonged exposure to severe oxidative stress induced by 0.4 mmol/l hydrogen peroxide. High-glucose treatment also significantly increased megalin expression in HK-2 cells. Concurrent administration of the antioxidant N-acetyl-cysteine blocked the effects of high glucose on megalin expression. Furthermore, the hydrogen peroxide-induced increase in megalin expression was blocked by treatment with phosphatidylinositol 3-kinase and Akt inhibitors. Increase of phosphorylated Akt expression was also seen in the renal cortex of diabetic rats. Taken together, our results indicate that mild oxidative stress increases renal megalin expression through the phosphatidylinositol 3-kinase-Akt pathway in the normoalbuminuric stage of DM.

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Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

International Journal of Immunopathology and Pharmacology ◽

10.1177/205873920601900104 ◽

2006 ◽

Vol 19 (1) ◽

pp. 205873920601900 ◽

Cited By ~ 35

Author(s):

M. Bongrazio ◽

L. DA Silva-Azevedo ◽

E.C. Bergmann ◽

O. Baum ◽

B. Hinz ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Western Blot ◽

Dependent Manner ◽

Vascular Networks ◽

Thrombospondin 1 ◽

Matrix Bound ◽

Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

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Empagliflozin improves endothelial and cardiomyocyte function in human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Gα oxidation

Cardiovascular Research ◽

10.1093/cvr/cvaa123 ◽

2020 ◽

Cited By ~ 10

Author(s):

Detmar Kolijn ◽

Steffen Pabel ◽

Yanna Tian ◽

Mária Lódi ◽

Melissa Herwig ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Ejection Fraction ◽

Linear Regression Analysis ◽

Lipid Peroxide ◽

Dependent Manner ◽

Preserved Ejection Fraction ◽

Human Heart Failure ◽

Stress Dependent

Abstract Aims Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). Methods and results The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. Conclusion Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO–sGC–cGMP–cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

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Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78-Loaded Extracellular Vesicles

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.315506 ◽

2020 ◽

Author(s):

Malgorzata Furmanik ◽

Rick van Gorp ◽

Meredith Whitehead ◽

Sadia Ahmad ◽

Jayanta Bordoloi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Er Stress ◽

Vascular Calcification ◽

Bone Mineralization ◽

Extracellular Vesicle ◽

Dependent Manner ◽

The Matrix ◽

Stress Dependent

Objective: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2, Osterix, ALP, BSP, and OPG), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3. EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. Conclusions: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

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Characterization of Brassica rapa RAP2.4-Related Proteins in Stress Response and as CUL3-Dependent E3 Ligase Substrates

Cells ◽

10.3390/cells8040336 ◽

2019 ◽

Vol 8 (4) ◽

pp. 336 ◽

Cited By ~ 2

Author(s):

Sutton Mooney ◽

Raed Al-Saharin ◽

Christina M. Choi ◽

Kyle Tucker ◽

Chase Beathard ◽

...

Keyword(s):

Brassica Rapa ◽

Stress Responses ◽

Expression Patterns ◽

Economic Value ◽

E3 Ligase ◽

26S Proteasome ◽

Dependent Manner ◽

E3 Ligases ◽

Subsequent Degradation ◽

Stress Dependent

The turnip Brassica rapa has important economic value and represents a good model system to study gene function in crop plants. ERF/AP2 transcription factors are a major group of proteins that are often involved in regulating stress-responses and developmental programs. Some ERF/AP2 proteins are targets of CULLIN3-based E3 ligases that use BTB/POZ-MATH proteins as substrate receptors. These receptors bind the transcription factor and facilitate their ubiquitylation and subsequent degradation via the 26S proteasome. Here, we show tissue and stress-dependent expression patterns for three Brassica rapa ERF/AP2 proteins that are closely related to Arabidopsis thaliana AtRAP2.4. Cloning of the Brassica genes showed that the corresponding proteins can assemble with a BPM protein and CULLIN3, and that they are instable in a 26S proteasome dependent manner. This work demonstrates the conserved nature of the ERF/AP2-CULLIN3-based E3 ligase interplay, and represents a first step to analyze their function in a commercially relevant crop plant.

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Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1

Biological Chemistry ◽

10.1515/bc.2011.040 ◽

2011 ◽

Vol 392 (5) ◽

Cited By ~ 28

Author(s):

Ingrid L. Cockburn ◽

Eva-Rachele Pesce ◽

Jude M. Pryzborski ◽

Michael T. Davies-Coleman ◽

Peter G.K. Clark ◽

...

Keyword(s):

Protein Aggregation ◽

Small Molecule ◽

Drug Target ◽

Molecular Probes ◽

Chaperone Activity ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Chaperone Function ◽

Hsp70 Chaperone ◽

Small Molecule Modulators

Abstract Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC50 values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

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Tumor-associated macrophages favor C26 murine colon carcinoma cell proliferation in an oxidative stress-dependent manner

Oncology Reports ◽

10.3892/or.2017.5466 ◽

2017 ◽

Vol 37 (4) ◽

pp. 2472-2480 ◽

Cited By ~ 8

Author(s):

Lavinia Luput ◽

Emilia Licarete ◽

Alina Sesarman ◽

Laura Patras ◽

Marius Costel Alupei ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Proliferation ◽

Colon Carcinoma ◽

Carcinoma Cell ◽

Dependent Manner ◽

Tumor Associated Macrophages ◽

Murine Colon Carcinoma ◽

Stress Dependent ◽

Murine Colon

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SOD1 overexpression prevents acute hyperglycemia-induced cerebral myogenic dysfunction: relevance to contralateral hemisphere and stroke outcomes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00321.2014 ◽

2015 ◽

Vol 308 (5) ◽

pp. H456-H466 ◽

Cited By ~ 10

Author(s):

Maha Coucha ◽

Weiguo Li ◽

Sherif Hafez ◽

Mohammed Abdelsaid ◽

Maribeth H. Johnson ◽

...

Keyword(s):

Ischemia Reperfusion ◽

Contralateral Side ◽

Neurological Deficits ◽

Myogenic Tone ◽

Dependent Manner ◽

Contralateral Hemisphere ◽

Acute Hyperglycemia ◽

Stroke Outcomes ◽

Novel Target ◽

Stress Dependent

Admission hyperglycemia (HG) amplifies vascular injury and neurological deficits in acute ischemic stroke, but the mechanisms remain controversial. We recently reported that ischemia-reperfusion (I/R) injury impairs the myogenic response in both hemispheres via increased nitration. However, whether HG amplifies contralateral myogenic dysfunction and whether loss of tone in the contralateral hemisphere contributes to stroke outcomes remain to be determined. Our hypothesis was that contralateral myogenic dysfunction worsens stroke outcomes after acute hyperglycemic stroke in an oxidative stress-dependent manner. Male wild-type or SOD1 transgenic rats were injected with saline or 40% glucose solution 10 min before surgery and then subjected to 30 min of ischemia/45 min or 24 h of reperfusion. In another set of animals ( n = 5), SOD1 was overexpressed only in the contralateral hemisphere by stereotaxic adenovirus injection 2–3 wk before I/R. Myogenic tone and neurovascular outcomes were determined. HG exacerbated myogenic dysfunction in contralateral side only, which was associated with infarct size expansion, increased edema, and more pronounced neurological deficit. Global and selective SOD1 overexpression restored myogenic reactivity in ipsilateral and contralateral sides, respectively, and enhanced neurovascular outcomes. In conclusion, our results show that SOD1 overexpression nullified the detrimental effects of HG on myogenic tone and stroke outcomes and that the contralateral hemisphere may be a novel target for the management of acute hyperglycemic stroke.

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tRNA-Derived Fragments Target the Ribosome and Function as Regulatory Non-Coding RNA inHaloferax volcanii

Archaea ◽

10.1155/2012/260909 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 128

Author(s):

Jennifer Gebetsberger ◽

Marek Zywicki ◽

Andrea Künzi ◽

Norbert Polacek

Keyword(s):

Gene Expression ◽

Regulatory Networks ◽

Ribosomal Subunit ◽

Fine Tuning ◽

Transferase Activity ◽

Dependent Manner ◽

Efficient Manner ◽

Stress Dependent

Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeonHaloferax volcaniidirectly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5′ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunitin vitroandin vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression inH. volcaniiunder environmental stress conditions probably by fine tuning the rate of protein production.

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