Revealing the elusive molecular biology of the vault RNA

Recently several novel and previously reported non-protein-coding RNAs (ncRNAs) have been identified to be upregulated upon Epstein-Barr virus (EBV) infection in human B-lymphocytes. A group of these significantly upregulated ncRNAs are called vault RNAs (vtRNAs). ,b Only about 5% of the total cellular vtRNAs are connected to the vault particle, the largest known ribonucleoprotein particle (RNP) in eukaryotic cells. However the function of this ncRNA family and moreover of the vault particle remains still rather unclear. Our previous findings suggest a link between EBV infection and vtRNA expression. Consequently we are interested which part of the viral genome is responsible for the upregulation and moreover which function the vtRNAs might possess during virus propagation. To address this question we have separately overexpressed specific EBV-encoded, latently expressed proteins in BL2-cells to determine the influence on the vault RNA levels. Thereby we identified one EBV-encoded protein, called Latent Membrane Protein 1 (LMP1), which significantly contributes to the vtRNA upregulation. We used LMP1 mutants to characterize the region of the protein and the responsible pathway for triggering the elevated vtRNA expression. Our results suggest that the NFkB- pathway might be involved in this process. To investigate a possible functional connection between the vtRNA and EBV infection, we have overexpressed vtRNA1-1 in BL41, a cell line usually not expressing this vault RNA. We show that overexpression of vtRNA1-1 leads to a better viral establishment and markedly protects cells from undergoing apoptosis. Knock-down of the major vault protein, the main component of the vault particle, had no effect on EBV infection and apoptosis resistance. Thus these results support the view that the observed phenotype is caused by the vtRNA rather than the vault particle.

The role of FUS in splicing regulation

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798

The FUS(s) about splicing

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Kidney disease in antiretroviral-naïve HIV-positive adults with high CD4 counts: prevalence and predictors of kidney disease at enrolment in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES HIV infection has been associated with an increased risk of chronic kidney disease (CKD). Little is known about the prevalence of CKD in individuals with high CD4 cell counts prior to initiation of antiretroviral therapy (ART). We sought to address this knowledge gap. METHODS We describe the prevalence of CKD among 4637 ART-naïve adults (mean age 36.8 years) with CD4 cell counts > 500 cells/μL at enrolment in the Strategic Timing of AntiRetroviral Treatment (START) study. CKD was defined by estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m(2) and/or dipstick urine protein ≥ 1+. Logistic regression was used to identify baseline characteristics associated with CKD. RESULTS Among 286 [6.2%; 95% confidence interval (CI) 5.5%, 6.9%] participants with CKD, the majority had isolated proteinuria. A total of 268 participants had urine protein ≥ 1+, including 41 with urine protein ≥ 2+. Only 22 participants (0.5%) had an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) , including four who also had proteinuria. Baseline characteristics independently associated with CKD included diabetes [adjusted odds ratio (aOR) 1.73; 95% CI 1.05, 2.85], hypertension (aOR 1.82; 95% CI 1.38, 2.38), and race/ethnicity (aOR 0.59; 95% CI 0.37, 0.93 for Hispanic vs. white). CONCLUSIONS We observed a low prevalence of CKD associated with traditional CKD risk factors among ART-naïve clinical trial participants with CD4 cell counts > 500 cells/μL.

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

Functional characterization of the trypanosome translational repressor SCD6

The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and tr

Assessing efficacy of different nucleos(t)ide backbones in NNRTI-containing regimens in the Swiss HIV Cohort Study.

BACKGROUND The most recommended NRTI combinations as first-line antiretroviral treatment for HIV-1 infection in resource-rich settings are tenofovir/emtricitabine, abacavir/lamivudine, tenofovir/lamivudine and zidovudine/lamivudine. Efficacy studies of these combinations also considering pill numbers, dosing frequencies and ethnicities are rare. METHODS We included patients starting first-line combination ART (cART) with or switching from first-line cART without treatment failure to tenofovir/emtricitabine, abacavir/lamivudine, tenofovir/lamivudine and zidovudine/lamivudine plus efavirenz or nevirapine. Cox proportional hazards regression was used to investigate the effect of the different NRTI combinations on two primary outcomes: virological failure (VF) and emergence of NRTI resistance. Additionally, we performed a pill burden analysis and adjusted the model for pill number and dosing frequency. RESULTS Failure events per treated patient for the four NRTI combinations were as follows: 19/1858 (tenofovir/emtricitabine), 9/387 (abacavir/lamivudine), 11/344 (tenofovir/lamivudine) and 45/1244 (zidovudine/lamivudine). Compared with tenofovir/emtricitabine, abacavir/lamivudine had an adjusted HR for having VF of 2.01 (95% CI 0.86-4.55), tenofovir/lamivudine 2.89 (1.22-6.88) and zidovudine/lamivudine 2.28 (1.01-5.14), whereas for the emergence of NRTI resistance abacavir/lamivudine had an HR of 1.17 (0.11-12.2), tenofovir/lamivudine 11.3 (2.34-55.3) and zidovudine/lamivudine 4.02 (0.78-20.7). Differences among regimens disappeared when models were additionally adjusted for pill burden. However, non-white patients compared with white patients and higher pill number per day were associated with increased risks of VF and emergence of NRTI resistance: HR of non-white ethnicity for VF was 2.85 (1.64-4.96) and for NRTI resistance 3.54 (1.20-10.4); HR of pill burden for VF was 1.41 (1.01-1.96) and for NRTI resistance 1.72 (0.97-3.02). CONCLUSIONS Although VF and emergence of resistance was very low in the population studied, tenofovir/emtricitabine appears to be superior to abacavir/lamivudine, tenofovir/lamivudine and zidovudine/lamivudine. However, it is unclear whether these differences are due to the substances as such or to an association of tenofovir/emtricitabine regimens with lower pill burden.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

Non-canonical actions of Nogo-A and its receptors

Nogo-A is a myelin associated protein and one of the most potent neurite growth inhibitors in the central nervous system. Interference with Nogo-A signaling has thus been investigated as therapeutic target to promote functional recovery in CNS injuries. Still, the finding that Nogo-A presents a fairly ubiquitous expression in many types of neurons in different brain regions, in the eye and even in the inner ear suggests for further functions besides the neurite growth repression. Indeed, a growing number of studies identified a variety of functions including regulation of neuronal stem cells, modulation of microglial activity, inhibition of angiogenesis and interference with memory formation. Aim of the present commentary is to draw attention on these less well-known and sometimes controversial roles of Nogo-A. Furthermore, we are addressing the role of Nogo-A in neuropathological conditions such as ischemic stroke, schizophrenia and neurodegenerative diseases.

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

Molecular mechanisms for the insulin sensitizing activity of rexinoids in skeletal muscle of diabetic (Db/Db) mice

Rexinoids are synthetic agonists for the retinoid X receptors (RXRs), a member of the nuclear receptor family of ligand-activated transcription factors. Rexinoids have been shown to lower serum glucose and insulin levels in animal models of type 2 diabetes. However the mechanisms that are responsible for the insulin-sensitizing action of rexinoids are largely unknown. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal and impaired insulin action in muscle is an important contributor to the pathophysiology of type 2 diabetes. Glucose transport is a rate-limiting step in glucose utilization. The goal of these studies is to examine the mechanisms of the anti-diabetic activity of rexinoids in skeletal muscle of diabetic db/db mice. The results we have obtained showed that treatment of db/db mice with rexinoids for two weeks resulted in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Insulin stimulates glucose transport in muscle via the regulation of both the insulin receptor substrate-1 (IRS-1)/Akt pathway and the Cbl-associated protein (CAP)/Cbl pathway. Rexinoids increased the insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation without effects on the activity of the CAP/Cbl pathway. The effects of rexinoids on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Serine 307 phosphorylation as well as qualitative and quantitative alterations in the fatty acyl-CoAs present within the muscle cells. In addition, rexinoids increased the expression of uncoupling protein 3 (UCP3) and activation of AMPK in diabetic muscle. This effect may also enhance the IRS-1/Akt signaling. We believe that it is the concerted activation of the IRS-1/Akt and AMPK signaling systems, a pharmacological mechanism that as far as we know, is unique to rexinoids, that results in the anti-diabetic effects of these drugs. Our results also suggest that the glucose-lowering mechanism of rexinoids is distinct from that of the thiazolidinediones (TZDs), peroxisome proliferator-activated receptor γ (PPARγ) agonists with well-characterized anti-diabetic activity. Rexinoids appear to represent a novel class of insulin sensitizers, with potential applications for the treatment of type 2 diabetes. ^

Persisting abdominal symptoms after travelers' diarrhea: Is there a genetic predisposition?

Functional gastrointestinal disorders (FGIDs) are defined as ailments of the mid or lower gastrointestinal tract which are not attributable to any discernable anatomic or biochemical defects.1 FGIDs include functional bowel disorders, also known as persisting abdominal symptoms (PAS). Irritable bowel syndrome (IBS) is one of the most common illnesses classified under PAS.2,3 This is the first prospective study that looks at the etiology and pathogenesis of post-infectious PAS in the context of environmental exposure and genetic susceptibility in a cohort of US travelers to Mexico. Our objective was to identify infectious, genetic and environmental factors that predispose to post infectious PAS. ^ Methods. This is a secondary data analysis of a prospective study on a cohort of 704 healthy North American tourists to Cuernavaca, Morelos and Guadalajara, Jalisco in Mexico. The subjects at risk for Travelers' diarrhea were assessed for chronic abdominal symptoms on enrollment and six months after the return to the US. ^ Outcomes. PAS was defined as disturbances of mid and lower gastrointestinal system without any known pathological or radiological abnormalities, or infectious, or metabolic causes. It refers to functional bowel disease, category C of functional gastrointestinal diseases as defined by the Rome II criterion. PAS was sub classified into Irritable bowel syndrome (IBS) and functional abdominal disease (FAD). ^ IBS is defined as recurrent abdominal pain or discomfort present at least 25% and associated with improvement with defecation, change in frequency and form of stool. FAD encompasses other abdominal symptoms of chronic nature that do not meet the criteria for IBS. It includes functional diarrhea, functional constipation, functional bloating: and unspecified bowel symptoms. ^ Results. Among the 704 travelers studied, there were 202 cases of PAS. The PAS cases included 175 cases of FAD and 27 cases of IBS. PAS was more frequent among subjects who developed traveler's diarrhea in Mexico compared to travelers who remained healthy during the short term visit to Mexico (52 vs. 38; OR = 1.8; CI, 1.3–2.5, P < 0.001). A statistically significant difference was noted in the mean age of subjects with PAS compared to healthy controls (28 vs. 34 yrs; OR = 0.97, CI, 0.95–0.98; P < 0.001). Travelers who experienced multiple episodes, a later onset of diarrhea in Mexico and passed greater numbers of unformed stools were more likely to be identified in PAS group at six months. Participants who developed TD caused by enterotoxigenic E.coli in Mexico showed a 2.6 times higher risk of developing FAD (P = 0.003). Infection with Providencia ssp. also demonstrated a greater risk to developing PAS. Subjects who sought treatment for diarrhea while in Mexico also displayed a significantly lower frequency of IBS at six months follow up (OR = 0.30; CI, 0.10–0.80; P = 0.02). ^ Forty six SNPs belonging to 14 genes were studied. Seven SNPs were associated with PAS at 6 months. These included four SNPs from the Caspase Recruitment Domain-Containing Protein 15 gene (CARD15), two SNPs from Surfactant Pulmonary-Associated Protein D gene (SFTPD) and one from Decay-Accelerating Factor For Complement gene (CD55). A genetic risk score (GRS) was composed based on the 7 SNPs that showed significant association with PAS. A 20% greater risk for PAS was noted for every unit increase in GRS. The risk increased by 30% for IBS. The mean GRS was high for IBS (2.2) and PAS (1.1) compared to healthy controls (0.51). These data suggests a role for these genetic polymorphisms in defining the susceptibility to PAS. ^ Conclusions. The study allows us to identify individuals at risk for developing post infectious IBS (PI-IBS) and persisting abdominal symptoms after an episode of TD. The observations in this study will be of use in developing measures to prevent and treat post-infectious irritable bowel syndrome among travelers including pre-travel counseling, the use of vaccines, antibiotic prophylaxis or the initiation of early antimicrobial therapy. This study also provides insights into the pathogenesis of post infectious PAS and IBS. (Abstract shortened by UMI.)^

Emerging roles for the double strand break repair protein 53BP1 in transcriptional regulation

Disruption of the mechanisms that regulate cell-cycle checkpoints, DNA repair, and apoptosis results in genomic instability and often leads to the development of cancer. In response to double stranded breaks (DSBs) as induced by ionizing radiation (IR), generated during DNA replication, or through immunoglobulin heavy chain (IgH) rearrangements in T and B cells of lymphoid origin, the protein kinases ATM and ATR are central players that activate signaling pathways leading to DSB repair. p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs) where it is recruited to or near sites of DNA damage. In addition to its well established role in DSB repair, multiple lines of evidence implicate 53BP1 in transcription which stem from its initial discovery as a p53 binding protein in a yeast two-hybrid screen. However, the mechanisms behind the role of 53BP1 in these processes are not well understood. ^ 53BP1 possesses several motifs that are likely important for its role in DSB repair including two BRCA1 C-terminal repeats, tandem Tudor domains, and a variety of phosphorylation sites. In addition to these motifs, we identified a glycine and arginine rich region (GAR) upstream of the Tudor domains, a sequence that is oftentimes serves as a site for protein arginine methylation. The focus of this project was to characterize the methylation of 53BP1 and to evaluate how methylation influenced the role of 53BP1 as a tumor suppressor. ^ Using a variety of biochemical techniques, we demonstrated that 53BP1 is methylated by the PRMT1 methyltransferase in vivo. Moreover, GAR methylation occurs on arginine residues in an asymmetric manner. We further show that sequences upstream of the Tudor domains that do not include the GAR stretch are sufficient for 53BP1 oligomerization in vivo. While investigating the role of arginine methylation in 53BP1 function, we discovered that 53BP1 associates with proteins of the general transcription apparatus as well as to other factors implicated in coordinating transcription with chromatin function. Collectively, these data support a role for 53BP1 in regulating transcription and provide insight into the possible mechanisms by which this occurs. ^

The role of AKT-mediated phosphorylation in suppression of MAD1 and the development of new approach in protein complex detection

MAX dimerization protein 1 (MAD1) is a basic-helix-loop-helix transcription factors that recruits transcription repressor such as HDAC to suppress target genes transcription. It antagonizes to MYC because the promoter binding sites for MYC are usually also serve as the binding sites for MAD1 so they compete for it. However, the mechanism of the switch between MYC and MAD1 in turning on and off of genes' transcription is obscure. In this study, we demonstrated that AKT-mediated MAD1 phosphorylation inhibits MAD1 transcription repression function. The association between MAD1 and its target genes' promoter is reduced after been phosphorylated by AKT; therefore, consequently, allows MYC to occupy the binding site and activates transcription. Mutation of such phosphorylation site abrogates the inhibition from AKT. In addition, functional assays demonstrated that AKT suppressed MAD1-mediated transcription repression of its target genes hTERT and ODC. Cell cycle and cell growth were also been released from inhibition by MAD1 in the presents of AKT. Taken together, our study suggests that MAD1 is a novel substrate of AKT and AKT-mediated MAD1 phosphorylation inhibits MAD1function; therefore, activates MAD1 target genes expression. ^ Furthermore, analysis of protein-protein interaction is indispensable for current molecular biology research, but multiplex protein dynamics in cells is too complicated to be analyzed by using existing biochemical methods. To overcome the disadvantage, we have developed a single molecule level detection system with nanofluidic chip. Single molecule was analyzed based on their fluorescent profile and their profiles were plotted into 2 dimensional time co-incident photon burst diagram (2DTP). From this 2DTP, protein complexes were characterized. These results demonstrate that the nanochannel protein detection system is a promising tool for future molecular biology. ^