Selective control of donor-acceptor Stenhouse adduct populations with non-selective stimuli

Variants in Autophagy Genes Affect Susceptibility to Both Crohn's Disease and Helicobacter pylori Infection

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.

Coregulator Codes of Transcriptional Regulation by Nuclear Receptors

The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases.

Retroperitoneal Fibrosis and Asbestosis—A Plausible Association?

Are nitric oxide synthases new players in the pathophysiology of fulminant hepatic failure?

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.

Assessment of Antibody Protection against Malaria Sporozoites Must Be Done by Mosquito Injection of Sporozoites

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

The multipotentiality and self-renewal ability of stem cells are controlled by intrinsic genetic pathways that are subject to regulation by extrinsic signals emanating from the stem cell niche. The stem cell niche provides a microenvironment composed of cellular structures or extracellular matrix in which stem cells are maintained as undifferentiated (1-6). The concept of “the stem cell niche” was first proposed in the studies of hematopoietic stem cell (HSC) (7), however in vivo evidence of its existence was first shown in the Drosophila germline stem cell (GSC) (8-10). Over the past several years there has been much progress made in identifing stem cell niches in different mammal tissues, including nerve, hair follicle, intestine, teeth and bone marrow (11-16). In this review our focus is on comparing Drosophila GSC-niches and mouse HSC-niches (two of the best characterized niches). By such comparison, we hope to provide some common principles of stem cell niches that will be useful in other tissue stem cell niche studies. In recent years remarkable progress has been made in identification and charaterization of the stem cell niches in invertebrate systems (10,17-22). In studies of GSCs in Drosophila, the ovary and testis provide relatively simple but elegant anatomic structures with few cell types and unique stem cell markers. These advantages facilitated identification of the cellular components of the stem cell niche and definition of the molecular basis of physical interaction between stem cells and their niches (23,24), and revealed key niche signals involved in stem cell regulation (18,25-31). In the murine hematopoietic system, HSCs have been well defined (32,33) but identification of the HSC niches is just beginning. This search has been hampered by the complexity of the bone marrow (BM) structure and cellular components, and by the lack of unique HSC markers or distinctive characteristics of BM stromal cells. Two HSC-niches have been proposed in murine BM, an osteoblastic niche and a vascular niche, in which osteoblasts and vascular endothelial cells have been respectively demonstrated as major components (34-41). A recent study suggested a population of reticular cells, named CXCL12-abundant-reticular (CAR) cells, expressing a high level of CXCL12 (also known as stromal cell-derived factor [SDF]-1 or pre-B-cell-growth-stimulating factor [PBSF]), are in contact with HSCs in both osteoblastic and vascular niches (42). Intriguingly, megakaryocytic progenitors attach to vascular endothelial cells for proliferation and maturation, suggesting they are different from HSCs that directly contact CAR cells instead of endothelial cells in the vascular niche (43). Early B cell progenitors also attach to CAR cells for their growth and maintenance (44). Shared use of the vascular niche by other hematopoietic progenitors, including myeloid progenitors, suggests it may play additional roles in regulation of lineage commitment and differentiation (43,45). Thus the niche model established in studies of Drosophila GSCs may provide insight for further investigate of the different BM niches and their roles in regulation HSCs.

The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.

Preoperative opioid use: a modifiable risk factor for poor postoperative outcomes