scholarly journals Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1497
Author(s):  
Moses New-Aaron ◽  
Paul G. Thomes ◽  
Murali Ganesan ◽  
Raghubendra Singh Dagur ◽  
Terrence M. Donohue ◽  
...  
Keyword(s):  
Gene Activation ◽  
Cytochrome P450 2E1 ◽  
Intrinsic Apoptosis ◽  
Second Hit ◽  
Protein Levels ◽  
Lysosome Biogenesis ◽  
Galectin 3 ◽  
Hiv Exposed ◽  
Generating System

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis–transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

scholarly journals Influence of protein (human galectin-3) design on aspects of lectin activity

2020 ◽  
Vol 154 (2) ◽  
pp. 135-153 ◽  
Author(s):  
Gabriel García Caballero ◽  
Donella Beckwith ◽  
Nadezhda V. Shilova ◽  
Adele Gabba ◽  
Tanja J. Kutzner ◽  
...  
Keyword(s):  
Protein Design ◽  
Full Range ◽  
Biological Information ◽  
The Novel ◽  
Modular Assembly ◽  
Binding Partners ◽  
Galectin 3 ◽  
Potential Applications ◽  
Similar Affinity

Abstract The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

scholarly journals Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Juan Zhao ◽  
Hui Li ◽  
Guangxin Chen ◽  
Lijun Du ◽  
Peiyan Xu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Checkpoint ◽  
Early Embryo ◽  
Vital Role ◽  
Control Group ◽  
Embryo Abortion ◽  
Protein Levels ◽  
Mitotic Checkpoint Complex

Abstract Background Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date. Methods In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting. Results statistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b. Conclusion These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

scholarly journals Single Cell Profiling of Acute Kidney Injury Reveals Novel Transcriptional Signatures, Mixed Identities and Epithelial-to-Stromal Crosstalk

2019 ◽  
Author(s):  
Valeria Rudman-Melnick ◽  
Mike Adam ◽  
Andrew Potter ◽  
Saagar M. Chokshi ◽  
Qing Ma ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Single Cell ◽  
Stromal Cells ◽  
Gene Activation ◽  
Kidney Injury ◽  
Rapid Decline ◽  
Renal Tubules ◽  
Developmental Gene ◽  
Single Cell Profiling

SummaryAcute kidney injury (AKI) is a rapid decline of renal function, with an incidence of up to 67% of intensive care unit patients. Current treatments are merely supportive, emphasizing the need for deeper understanding that could lead to improved therapies. We used single cell RNA sequencing, in situ hybridization and protein expression analyses to create comprehensive renal cell specific transcriptional profiles of multiple AKI stages. We revealed that AKI induces marked dedifferentiation, renal developmental gene activation and mixed identities in injured renal tubules. Moreover, we identified potential pathologic crosstalk between epithelial and stromal cells, and several novel genes involved in AKI. We also demonstrated the definitive effects of age on AKI outcome, and showed that renal developmental genes hold a potential as novel AKI markers. Moreover, our study provides the resource power which will aid in unraveling the molecular genetics of AKI.

Expression of bcl-2 and bax in regenerating rat renal tubules following ischemic injury

1997 ◽  
Vol 272 (5) ◽  
pp. F640-F647 ◽  
Author(s):  
D. P. Basile ◽  
H. Liapis ◽  
M. R. Hammerman
Keyword(s):  
Proximal Tubule ◽  
Ischemic Injury ◽  
Fold Increase ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Bax Protein ◽  
Adult Kidney ◽  
Predictable Pattern

To define potential roles for bcl-2 and bax in adult kidney as regulators of regeneration, their expressions were characterized postischemic injury. A 2.1-fold increase in levels of renal bcl-2 mRNA occurred within 24 h of injury relative to levels in kidney of sham-operated control rats. The levels of bcl-2 mRNA remained elevated for 3 days but returned to baseline by day 5 postischemia. In situ hybridization of kidneys from sham-operated rats demonstrated faint expression of bcl-2 mRNA localized diffusely throughout the nephron. After renal injury, the expression of bcl-2 mRNA was markedly enhanced in regenerating proximal tubule cells relining the basement membrane. Immunohistochemistry showed a similar localization for bcl-2 protein. Levels of bax mRNA in kidney were elevated beginning at 24 h postischemia and remained elevated for 7 days postinjury. Bax mRNA and bax protein were colocalized to regenerating proximal tubules postischemia and were prominently expressed in papillary proliferations. We conclude that the expressions of bcl-2 and bax in kidney are enhanced in a predictable pattern following acute ischemic injury. Our findings suggest that these regulators of apoptosis play key roles in the process of repair of the damaged proximal tubule postischemia.

scholarly journals A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets

2007 ◽  
Vol 204 (7) ◽  
pp. 1571-1582 ◽  
Author(s):  
Ronit Pasvolsky ◽  
Sara W. Feigelson ◽  
Sara Sebnem Kilic ◽  
Amos J. Simon ◽  
Guy Tal-Lapidot ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Leukocyte Adhesion ◽  
Splice Junction ◽  
Integrin Activation ◽  
Leukocyte Adhesion Deficiency ◽  
Protein Levels ◽  
Guanine Exchange Factor ◽  
Β3 Integrin ◽  
Inside Out

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

scholarly journals Galectin-1 and galectin-3 in the human intervertebral disc: evidence for different localization patterns and gene activation profiles

2019 ◽  
Vol 27 ◽  
pp. S464-S465
Author(s):  
S. Toegel ◽  
K. Kinslechner ◽  
M. Elshamly ◽  
D. Weinmann ◽  
S.M. Walzer ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Gene Activation ◽  
Galectin 3

scholarly journals Engineered algae: A novel oxygen-generating system for effective treatment of hypoxic cancer

2020 ◽  
Vol 6 (21) ◽  
pp. eaba5996 ◽  
Author(s):  
Yue Qiao ◽  
Fei Yang ◽  
Tingting Xie ◽  
Zhen Du ◽  
Danni Zhong ◽  
...  
Keyword(s):  
Proof Of Concept ◽  
Tumor Cell Apoptosis ◽  
Cancer Management ◽  
Novel Approach ◽  
Hypoxic Environment ◽  
Sequential Combination ◽  
Hypoxic Tumor ◽  
Local Oxygen ◽  
Generating System

Microalgae, a naturally present unicellular microorganism, can undergo light photosynthesis and have been used in biofuels, nutrition, etc. Here, we report that engineered live microalgae can be delivered to hypoxic tumor regions to increase local oxygen levels and resensitize resistant cancer cells to both radio- and phototherapies. We demonstrate that the hypoxic environment in tumors is markedly improved by in situ–generated oxygen through microalgae-mediated photosynthesis, resulting in notably radiotherapeutic efficacy. Furthermore, the chlorophyll from microalgae produces reactive oxygen species during laser irradiation, further augmenting the photosensitizing effect and enhancing tumor cell apoptosis. Thus, the sequential combination of oxygen-generating algae system with radio- and phototherapies has the potential to create an innovative treatment strategy to improve the outcome of cancer management. Together, our findings demonstrate a novel approach that leverages the products of photosynthesis for treatment of tumors and provide proof-of-concept evidence for future development of algae-enhanced radio- and photodynamic therapy.

scholarly journals Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury “stress response”

2012 ◽  
Vol 303 (1) ◽  
pp. F139-F148 ◽  
Author(s):  
Richard A. Zager ◽  
Anitha Vijayan ◽  
Ali C. M. Johnson
Keyword(s):  
Acute Kidney Injury ◽  
Proximal Tubule ◽  
Ischemia Reperfusion ◽  
Gene Activation ◽  
Kidney Injury ◽  
Normal Subjects ◽  
Gene Induction ◽  
Urine Samples ◽  
Protein Levels ◽  
Neutrophil Gelatinase

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4–72 h or 1–3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ∼10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (∼12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.

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