Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells

Aims: Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. Methods: Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. Results: Oxidative stress induced more apoptosis in HK2s than MDCKs (p<0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p<0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p<0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p<0.01), and p-cav1 was induced by treatment. Conclusion: Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress. © 2008 Royal College of Pathologists of Australasia.

Tubule detection in testis images using boundary weighting and circular shortest path

In studies of germ cell transplantation, measureing tubule diameters and counting cells from different populations using antibodies as markers are very important. Manual measurement of tubule sizes and cell counts is a tedious and sanity grinding work. In this paper, we propose a new boundary weighting based tubule detection method. We first enhance the linear features of the input image and detect the approximate centers of tubules. Next, a boundary weighting transform is applied to the polar transformed image of each tubule region and a circular shortest path is used for the boundary detection. Then, ellipse fitting is carried out for tubule selection and measurement. The algorithm has been tested on a dataset consisting of 20 images, each having about 20 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually. © 2013 IEEE.

The promoter of cell growth- and RNA protection-associated SND1 gene is activated by endoplasmic reticulum stress in human hepatoma cells

Background: Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat- shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells. Results: We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5'-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real- time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress- inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor. Conclusion: Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcRI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a PGE2-Dependent Mechanism and the Involvement of a Proximal cAMP-Responsive Element.

Abstract The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is upregulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 over-expression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2-fold and 3.2-fold higher, respectively, in Cox-2 over-expressing cells (PCXII) vs their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E2 (1u�­M), mimicked this effect in PCMT cells, while treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nM) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neurone specific enolase (NSE) were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA upregulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E2-dependent upregulation required a proximal cAMP-responsive element (CRE; -71 - -64 bp). This study is the first to demonstrate that Cox-2 upregulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is upregulated.

Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

DNA methylation profiling in cell models of diabetic nephropathy

We have previously identified differentially expressed genes in cell models of diabetic nephropathy and renal biopsies. Here we have performed quantitative DNA methylation profiling in cell models of diabetic nephropathy. Over 3,000 CpG units in the promoter regions of 192 candidate genes were assessed in unstimulated human mesangial cells (HMCs) and proximal tubular epithelial cells (PTCs) compared to HMCs or PTCs exposed to appropriate stimuli. A total of 301 CpG units across 38 genes (similar to 20%) were identified as differentially methylated in unstimulated HMCs versus PTCs. Analysis of amplicon methylation values in unstimulated versus stimulated cell models failed to demonstrate a >20% difference between amplicons. In conclusion, our results demonstrate that specific DNA methylation signatures are present in HMCs and PTCs, and standard protocols for exposure of renal cells to stimuli that alter gene expression may be insufficient to replicate possible alterations in DNA methylation profiles in diabetic nephropathy.

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

Acute Upper Limb Ischemia, a Rare Presentation of Giant Cell Arteritis

Giant cell arteritis (GCA) is a systemic large vessel vasculitis, with extracranial arterial involvement described in 10-15% of cases, usually affecting the aorta and its branches. Patients with GCA are more likely to develop aortic aneurysms, but these are rarely present at the time of the diagnosis. We report the case of an 80-year-old Caucasian woman, who reported proximal muscle pain in the arms with morning stiffness of the shoulders for eight months. In the previous two months, she had developed worsening bilateral arm claudication, severe pain, cold extremities and digital necrosis. She had no palpable radial pulses and no measurable blood pressure. The patient had normochromic anemia, erythrocyte sedimentation rate of 120 mm/h, and a negative infectious and autoimmune workup. Computed tomography angiography revealed concentric wall thickening of the aorta extending to the aortic arch branches, particularly the subclavian and axillary arteries, which were severely stenotic, with areas of bilateral occlusion and an aneurysm of the ascending aorta (47 mm). Despite corticosteroid therapy there was progression to acute critical ischemia. She accordingly underwent surgical revascularization using a bilateral carotid-humeral bypass. After surgery, corticosteroid therapy was maintained and at six-month follow-up she was clinically stable with reduced inflammatory markers. GCA, usually a chronic benign vasculitis, presented exceptionally in this case as acute critical upper limb ischemia, resulting from a massive inflammatory process of the subclavian and axillary arteries, treated with salvage surgical revascularization.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.