883 research outputs found
Drosophila Bruce Can Potently Suppress Rpr- and Grim-Dependent but Not Hid-Dependent Cell Death
Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2) 1, 2. BRUCE upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs [2]. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Here, we demonstrate, using gain-of-function and deletion alleles, that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that dBruce can regulate cell death at a novel point
Sulfation degree of glycosaminoglycans triggers distinct cytoskeleton organisation in mesenchymal stem cells
Glycosaminoglycans (GAGs) comprise the closest cellular environment: they are building
elements of the ECM and can be also found on cells surface. Their biological activity depends
on several parameters among which the negative charge is of prime importance[1]. This
charge is generally associated with the presence of sulfate groups (-OSO3H). Sulfation is
a dynamic modification: it can occur at various positions within the glycan and different
sulfation patterns have been identified for the same organs and cells during their
development. However, the mechanisms of coding and transferring information by
these functionalities are not yet complete understood, mainly because of (i)the complex
physiological microenvironment in which GAGs interactions occur and (ii)the inability to access
homogeneous GAGs[2].
In this work, we propose model surfaces bearing GAGs with different sulfation degree as
platform to investigate the pathways by which mesenchymal stem cells (MSCs) sense and
respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this
study: hyaluronic acid (HA) because it is the only non-sulfated glycan and heparin (HEP) as it is
the GAG with the highest degree of sulfation. To obtain a larger range of sulfation degrees, we
have also prepared a synthetic analogue of HA with a sulfation degree of 1.4 (sHA). All these
GAGs were covalently bonded to aminothiols deposited on gold surfaces. MSCs, both from
bone marrow and adipose tissue, adhered well to all surfaces. Formation of focal adhesions
was observed after only 1h of culture for bone marrow derived MSCs regardless the used
substrate. The presence of –OSO3H groups induced different morphology and cytoskeleton
organisation: formation of longer filopodia and well pronounced actin fibers were visible for
the MSCs from both sources. Moreover, cells were more spread after 24h in contact with –
OSO3H containing surfaces. Cells behaved similarly on both sulfated surfaces (sHA and HEP)
and differences in cell morphology were less obvious: higher sulfation degree induced less
lamellipodia formation while filopodia number and length increased.
In summary, the present study provides evidence that sulfation degree of GAGs triggers
distinct cytoskeleton organisation in mesenchymal stem cells that may be related with the
differentiation of those cells. However, further studies at the molecular level about the exact
mechanism of these processes need to be carried out
Epigenetic effects of metformin: From molecular mechanisms to clinical implications
There is a growing body of evidence that links epigenetic modifications to type 2 diabetes. Researchers have more recently investigated effects of commonly used medications, including those prescribed for diabetes, on epigenetic processes. This work reviews the influence of the widely used antidiabetic drug metformin on epigenomics, microRNA levels and subsequent gene expression, and potential clinical implications. Metformin may influence the activity of numerous epigenetic modifying enzymes, mostly by modulating the activation of AMP-activated protein kinase (AMPK). Activated AMPK can phosphorylate numerous substrates, including epigenetic enzymes such as histone acetyltransferases (HATs), class II histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), usually resulting in their inhibition; however, HAT1 activity may be increased. Metformin has also been reported to decrease expression of multiple histone methyltransferases, to increase the activity of the class III HDAC SIRT1 and to decrease the influence of DNMT inhibitors. There is evidence that these alterations influence the epigenome and gene expression, and may contribute to the antidiabetic properties of metformin and, potentially, may protect against cancer, cardiovascular disease, cognitive decline and aging. The expression levels of numerous microRNAs are also reportedly influenced by metformin treatment and may confer antidiabetic and anticancer activities. However, as the reported effects of metformin on epigenetic enzymes act to both increase and decrease histone acetylation, histone and DNA methylation, and gene expression, a significant degree of uncertainty exists concerning the overall effect of metformin on the epigenome, on gene expression, and on the subsequent effect on the health of metformin users
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IDOL regulates systemic energy balance through control of neuronal VLDLR expression.
Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity
Tumour Suppressive Function and Modulation of Programmed Cell Death 4 (PDCD4) in Ovarian Cancer
Background: Programmed cell death 4 (PDCD4), originally identified as the neoplastic transformation inhibitor, was attenuated in various cancer types. Our previous study demonstrated a continuous down-regulation of PDCD4 expression in the sequence of normal-borderline-malignant ovarian tissue samples and a significant correlation of PDCD4 expression with disease-free survival. The objective of the current study was to further investigate the function and modulation of PDCD4 in ovarian cancer cells. Principal Findings: We demonstrated that ectopic PDCD4 expression significantly inhibited cell proliferation by inducing cell cycle arrest at G1 stage and up-regulation of cell cycle inhibitors of p27 and p21. Cell migration and invasion were also inhibited by PDCD4. PDCD4 over-expressing cells exhibited elevated phosphatase and tensin homolog (PTEN) and inhibited protein kinase B (p-Akt). In addition, the expression of PDCD4 was up-regulated and it was exported to the cytoplasm upon serum withdrawal treatment, but it was rapidly depleted via proteasomal degradation upon serum re-administration. Treatment of a phosphoinositide 3-kinase (PI3K) inhibitor prevented the degradation of PDCD4, indicating the involvement of PI3K-Akt pathway in the modulation of PDCD4. Conclusion: PDCD4 may play a critical function in arresting cell cycle progression at key checkpoint, thus inhibiting cell proliferation, as well as suppressing tumour metastasis. The PI3K-Akt pathway was implied to be involved in the regulatio
Novel mutations expand the clinical spectrum of DYNC1H1-associated spinal muscular atrophy
OBJECTIVE
To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene.
METHODS
Patients with a phenotype suggestive of a motor, non-length-dependent neuronopathy predominantly affecting the lower limbs were identified at participating neuromuscular centers and referred for targeted sequencing of DYNC1H1.
RESULTS
We report a cohort of 30 cases of SMA-LED from 16 families, carrying mutations in the tail and motor domains of DYNC1H1, including 10 novel mutations. These patients are characterized by congenital or childhood-onset lower limb wasting and weakness frequently associated with cognitive impairment. The clinical severity is variable, ranging from generalized arthrogryposis and inability to ambulate to exclusive and mild lower limb weakness. In many individuals with cognitive impairment (9/30 had cognitive impairment) who underwent brain MRI, there was an underlying structural malformation resulting in polymicrogyric appearance. The lower limb muscle MRI shows a distinctive pattern suggestive of denervation characterized by sparing and relative hypertrophy of the adductor longus and semitendinosus muscles at the thigh level, and diffuse involvement with relative sparing of the anterior-medial muscles at the calf level. Proximal muscle histopathology did not always show classic neurogenic features.
CONCLUSION
Our report expands the clinical spectrum of DYNC1H1-related SMA-LED to include generalized arthrogryposis. In addition, we report that the neurogenic peripheral pathology and the CNS neuronal migration defects are often associated, reinforcing the importance of DYNC1H1 in both central and peripheral neuronal functions
System-level studies of a cell-free transcription-translation platform for metabolic engineering
Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development
Effects of partner proteins on BCA2 RING ligase activity
Abstract
Background
BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.
Methods
Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.
Results
Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = \u3c 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.
Conclusions
The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression
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