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Identification of Molecular Determinants that Shift Co- and Post-Translational N-Glycosylation Kinetics in Type I Transmembrane Peptides: A Dissertation

Mon, 06/16/2014 - 4:00pm

Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation.

To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini.

In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites.

Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates.

Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.

Building the Future: Rejuvenating Librarianship

Mon, 06/16/2014 - 3:23pm

There is often a gap between what new library school graduates know and what is needed to navigate the first professional job. Additionally, the field of librarianship in general is growing and changing rapidly, requiring a diverse set of complex skills in new roles. It is essential to rethink how we train the next generation of librarians, while accepting and incorporating the unique insights new graduates may have. How can institutions help shape the next generation, while building skills that will be beneficial to all?

These challenges are being faced and addressed by an innovative library fellows program at the Lamar Soutter Library at the University of Massachusetts Medical School. This program is designed to foster the next generation of medical librarians by providing a 2-year experience for newly graduated library science students, emphasizing hands-on learning and research into topics of information management and the various complex roles within librarianship. An original curriculum has been developed incorporating training, professional development, mentorship, and research with the library as the learning laboratory. Curriculum components focus on librarianship foundations as well as rotations within core library functional areas.

Creating a culture of research within the library as a whole has also become a priority. Library fellows will conduct research designed to provide a self-directed course of study and investigation, ultimately with the goal of publishing the results. The research experience will expose the fellows to the research process and interpretation of results for decision-making in a library environment, as well as encouraging continued contribution to the field. This is facilitated by a research project database to which all staff can contribute and from which all can draw for ongoing academic discussion.

This presentation will provide a description and evaluation of the project to date, with successes, challenges, suggestions, and lessons learned discussed. It will look at the organizational changes that necessitated and facilitated the structural changes surrounding this program and the resulting effect on staff and operations. Administrators and creators of the program, as well as the first cohort of fellows, will present their perspectives.

Dramatic changes in how we structure libraries and librarianship are necessary to sustain and grow. These changes require rejecting old service models, rethinking our roles, redoing our professional identity and rejuvenating ourselves and our libraries. By changing the way we develop the knowledge and skills of new professionals, those who will be driving librarianship in the future, we strengthen all members of the profession and create a better, stronger foundation for librarianship.

Building the Future: Rejecting, Rethinking, Redoing, Rejuvenating

Mon, 06/16/2014 - 1:57pm

Objectives: Traditional library work is spiraling downward. Health sciences librarians are taking on new roles such as embedded librarians or research data informationists. Simultaneously, institutionally mandated budget cuts force the question, "How do we maintain mission-critical work within our budget?" Survival means rejecting old service models, rethinking our roles, redoing our professional identity, and rejuvenating ourselves and our libraries.

Methods: The Library Fellows Program at the University of Massachusetts (UMass) Medical School is one response to the challenges we are facing. The fellows program, designed to foster the next generation of medical librarians, provides a two-year experience for newly graduated library science students, emphasizing hands-on learning and research into topics of information management and medical librarianship. This innovative curriculum incorporates training, professional development, mentorship, and research with the library as the learning laboratory. Curriculum components focus on medical librarianship foundations as well as rotations within core library functional areas. This paper serves as a project description and evaluation. It discusses organizational changes that necessitated and facilitated the structural changes surrounding this program and the resulting effect on staff and operations. The midpoint success of the program is determined and reported, with recommendations and future considerations.

Results/Conclusions: In early 2013, management at Lamar Soutter Library (LSL) planned organizational changes necessary to meet strategic initiatives and continue supporting the medical school's mission in the face of severe budget constraints. The final plan resulted in discontinuation of many traditional library activities, elimination of staff that supported those activities, and, ultimately, the development of the FELLOWS PROGRAM. In September 2013, three task forces were created to develop an implementation plan. A search committee was formed to begin the process of hiring three fellows. The Curriculum Task Force was charged with structuring the two-year fellowship program. The curriculum developed includes rotations through library departments, in-depth reference experience, expert searching training, structured projects, and performing research. The Reference Services Task Force was charged with developing a new reference model to replace the current triage and pager model. The Research Task Force was charged with laying the groundwork for creating a research environment in the library. With outside consultation, LSL developed a detailed evaluation plan. The program is in its eighth month. Modifications and refinements are being made as the first cohort experiences the program. The program has led to a redefinition of librarianship and a new professional identity based on a culture of achievement, research, and reflection.

Electronic Bedside Documentation and Nurse-Patient Communication: A Dissertation

Mon, 06/16/2014 - 1:41pm

Nurses are often the first members of the health care team with whom patients interact. The initial impression of the nurses’ receptiveness to the patients’ needs influences the patients’ views of their overall care. Researchers have suggested that understanding communication between individuals can provide the human link, or social element, to the successful implementation and use of electronic health records, including documentation (Lanham, Leykum, & McDaniel, 2012). Zadvinskis, Chipps, and Yen (2014) identified that the helpful features of bedside documentation systems were offset by the mismatch between the system and nurse’s workflow. The purpose of this micro-ethnography study was to explore the culture of nurse-patient interaction associated with electronic documentation at the bedside. Data were collected through passive participant observation, audio-taping of the nurse-patient interactions, and informal and semi-structured interviews with the nurses. A total of twenty-six observations were conducted on three nursing units at an urban healthcare facility in New England. These three units were occupied by similar patient populations and all patients required cardiac monitoring. Three themes consistently emerged from qualitative data analysis: the nurses paused during verbal communication, the nurses played a game of tag between the patient and the computer, and the nurses performed automatic or machine-like actions. The participants described these themes in the informal and semi-structured interviews. The nurses’ actions were observed during passive participant observation, and the audio-taped interactions supported these themes. Understanding the adaptation of caregiving necessitated by bedside electronic documentation will have a positive impact on developing systems that interface seamlessly with the nurses’ workflow and encourage patients’ active participation in their care.

Examining Change in Symptoms of Depression, Anxiety, and Stress in Adults after Treatment of Chronic Cough: A Dissertation

Mon, 06/16/2014 - 1:40pm

Background: Chronic cough is a common health problem with variable success rates to standardized treatment. Psychologic symptoms of depression, anxiety, and stress have been reported in association with chronic cough. The purpose of this study was to examine changes in the psychologic symptoms of depression, anxiety, and stress in adults with chronic cough 3 months after management using the ACCP cough treatment guidelines.

Methods: This study used a descriptive longitudinal observation design. The major tenets associated with the Theory of Unpleasant Symptoms were examined. Intervention fidelity to the study components was measured.

Results: A sample of 80 consecutive patients with chronic cough of greater than 8 weeks duration was recruited from one cough specialty clinic. Mean age of subjects was 58.54 years; 68.7% were female; 98.7% were white, and 97.5% were non-smokers. Mean cough duration was 85.99 months and mean cough severity was 6.11 (possible 0 –10; higher scores equal greater cough severity). Cough severity improved post treatment (n=65, M=2.32, (SE =.291), t (64) =7.98, p=.000); cough-specific quality-of-life also improved (n=65, M=9.17, (SE=1.30), t (64) =7.02, p=.000). Physiologic (urge-to-cough r=.360, ability to speak r=.469) and psychologic factors (depression r=.512, anxiety r=.507, stress r=.484) were significantly related to cough-specific quality-of-life and to cough severity (urge-to-cough r=.643, ability to speak r=.674 and depression r=.356, anxiety r=.419, stress r=.323) (all r, p=.01); social support and number of diagnoses were not related to either variable. Those experiencing greater financial strain had worse cough severity. Women, those experiencing financial strain, and those taking self-prescribed therapy had worse cough-specific quality-of-life. Intervention fidelity to the study plan was rated as high according to observation, participant receipt, and patient/physician concordance. Qualitative review identified potential areas of variability with intervention fidelity.

Conclusions: By measuring the factors related to the major tenets of the Theory of Unpleasant Symptoms, this theory has helped to explain why those with chronic cough may have symptoms of depression, anxiety, and stress and why these symptoms improve as cough severity and cough-specific quality-of-life improve. Moreover, by measuring intervention fidelity, it may be possible to determine why cough guidelines may not be yielding consistently favorable results.

Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications

Wed, 06/11/2014 - 3:30pm

Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca(2+) binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca(2+) binding. Cryo-electron microscopy and single-particle image reconstruction of Ca(2+)-regulated (scallop) filaments reveals a helical array of myosin head-pair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation--involving phosphorylation or Ca(2+) binding--share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca(2+)-binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca(2+)-regulated myosin.

Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function

Wed, 06/11/2014 - 3:30pm

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C((t/t))). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C((t/t)) mice. Slow and fast skeletal muscles in cMyBP-C((t/t)) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C((t/t)) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

Different head environments in tarantula thick filaments support a cooperative activation process

Wed, 06/11/2014 - 3:30pm

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Wed, 06/11/2014 - 3:30pm

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state

Wed, 06/11/2014 - 3:30pm

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 A resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes. reserved.

Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C

Wed, 06/11/2014 - 3:30pm

Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to beta-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart.

BRG1, a SWI/SNF chromatin remodeling enzyme ATPase, is required for maintenance of nuclear shape and integrity

Wed, 06/11/2014 - 12:15pm

We recently reported that reducing the levels of BRG1, the catalytic subunit of mammalian SWI/SNF chromatin remodeling enzymes, induces alterations in nuclear shape in a breast epithelial cell line. Immunostaining the BRG1 knockdown cells with nuclear lamina antibodies revealed a significantly increased frequency of grooves, or invaginations, in the nuclei. Disruption of each of the major cytoplasmic filament systems (actin, tubulin and cytokeratins) had no impact on the BRG1-dependent changes in nuclear shape, indicating that the observed changes in nuclear morphology are unlikely to be a result of alterations in the integrity of the nuclear-cytoplamic contacts in the cell. We propose that the BRG1-dependent nuclear shape changes reflect a role for the chromatin remodeling enzyme in maintaining the structural integrity of the nucleus via global regulation of chromatin structure and dynamics within the nucleus.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation

Wed, 06/11/2014 - 12:15pm

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPalpha-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

The PPARgamma locus makes long-range chromatin interactions with selected tissue-specific gene loci during adipocyte differentiation in a protein kinase A dependent manner

Wed, 06/11/2014 - 12:15pm

Differentiation signaling results in reprogramming of cellular gene expression that leads to morphological changes and functional specialization of a precursor cell. This global change in gene expression involves temporal regulation of differentiation-specific genes that are located throughout the genome, raising the idea that genome structure may also be re-organized during cell differentiation to facilitate regulated gene expression. Using in vitro adipocyte differentiation as a model, we explored whether gene organization within the nucleus is altered upon exposure of precursor cells to signaling molecules that induce adipogenesis. The peroxisome proliferator-activated receptor gamma (PPARgamma) nuclear hormone receptor is a master determinant of adipogenesis and is required for adipose differentiation. We utilized the chromosome conformation capture (3C) assay to determine whether the position of the PPARgamma locus relative to other adipogenic genes is changed during differentiation. We report that the PPARgamma2 promoter is transiently positioned in proximity to the promoters of genes encoding adipokines and lipid droplet associated proteins at 6 hours post-differentiation, a time that precedes expression of any of these genes. In contrast, the PPARgamma2 promoter was not in proximity to the EF1alpha promoter, which drives expression of a constitutively active, housekeeping gene that encodes a translation elongation factor, nor was the PPARgamma2 promoter in proximity to the promoter driving the expression of the C/EBPalpha regulatory protein. The formation of the long-range, intergenic interactions involving the PPARgamma2 promoter required the regulatory factor C/EBPbeta, elevated cyclic AMP (cAMP) levels, and protein kinase A (PKA) signaling. We conclude that genome organization is dynamically remodeled in response to adipogenic signaling, and we speculate that these transient inter-genic interactions may be formed for the purposes of selecting some of the transcriptionally silent tissue-specific loci for subsequent transcriptional activation.

Developmental profile and sexually dimorphic expression of kiss1 and kiss1r in the fetal mouse brain

Wed, 06/11/2014 - 11:25am

The hypothalamic-pituitary-gonadal axis (HPG) is a complex neuroendocrine circuit involving multiple levels of regulation. Kisspeptin neurons play essential roles in controlling the HPG axis from the perspectives of puberty onset, oscillations of gonadotropin releasing hormone (GnRH) neuron activity, and the pre-ovulatory LH surge. The current studies focus on the expression of kisspeptin during murine fetal development using in situ hybridization (ISH), quantitative reverse transcription real-time PCR (QPCR), and immunocytochemistry. Expression of mRNA coding for kisspeptin (KISS1) and its receptor KISS1R was observed at embryonic (E) day 13 by ISH. At E13 and other later ages examined, Kiss1 signal in individual cells within the arcuate nucleus (ARC) appeared stronger in females than males. ISH examination of agonadal steroidogenic factor-1 (Sf1) knockout mice revealed that E17 XY knockouts (KO) resembled wild-type (WT) XX females. These findings raise the possibility that gonadal hormones modulate the expression of Kiss1 in the ARC prior to birth. The sex and genotype differences were tested quantitatively by QPCR experiments in dissected hypothalami from mice at E17 and adulthood. Females had significantly more Kiss1 than males at both ages, even though the number of cells detected by ISH was similar. In addition, QPCR revealed a significant difference in the amount of Kiss1 mRNA in Sf1 mice with WT XY mice expressing less than XY KO and XX mice of both genotypes. The detection of immunoreactive KISS1 in perikarya of the ARC at E17 indicates that early mRNA is translated to peptide. The functional significance of this early expression of Kiss1 awaits elucidation.

N-linked polylactosamine glycan synthesis is regulated by co-expression of beta3GnT2 and GCNT2

Wed, 06/11/2014 - 11:25am

Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galbeta1,4-GlcNAcbeta1,3)n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for beta1,4-galactosyltransferase (beta4GalT) and beta1,3-N-acetylglucosminyltransferase (beta3GnT). Because beta4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of eight known beta3GnT genes in mice. In the olfactory epithelium (OE), beta3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here, we show for the first time that the beta1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that co-express beta3GnT2 and PLN. This highly specific co-expression suggests that GCNT2 and beta3GnT2 function cooperatively in PLN synthesis. In support of this, beta3GnT2 and GCNT2 co-transfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with beta3GnT2 to determine PLN levels in olfactory neurons by regulating beta1,6-branches that promote PLN extension.

DNA-damage-induced nuclear export of precursor microRNAs is regulated by the ATM-AKT pathway

Wed, 06/11/2014 - 11:15am

Expression of microRNAs (miRNAs) involves transcription of miRNA genes and maturation of the primary transcripts. Recent studies have shown that posttranscriptional processing of primary and precursor miRNAs is induced after DNA damage through regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP. However, little is known about the regulation of nuclear export of pre-miRNAs in the DNA-damage response, a critical step in miRNA maturation. Here, we show that nuclear export of pre-miRNAs is accelerated after DNA damage in an ATM-dependent manner. The ATM-activated AKT kinase phosphorylates Nup153, a key component of the nucleopore, leading to enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings define an important role of DNA-damage signaling in miRNA transport and maturation.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma

Wed, 06/11/2014 - 11:15am

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

Fidelity of Histone Gene Regulation is Obligatory for Genome Replication and Stability

Wed, 06/11/2014 - 11:15am

Fidelity of chromatin organization is crucial for normal cell cycle progression and perturbations in packaging of DNA may predispose to transformation. Histone H4 protein is the most highly conserved chromatin protein, required for nucleosome assembly with multiple histone H4 gene copies encoding identical protein. There is a long-standing recognition of the linkage of histone gene expression and DNA replication. A fundamental and unresolved question is the mechanism that couples histone biosynthesis with DNA replication and fidelity of cell cycle control. Here, we conditionally ablated the obligatory histone H4 transcription factor Hinfp to cause depletion of histone H4 in mammalian cells. Deregulation of histone H4 results in catastrophic cellular and molecular defects that lead to genomic instability. Histone H4 depletion increases nucleosome spacing, impedes DNA synthesis, alters chromosome complement, and creates replicative stress. Our study provides functional evidence that the tight coupling between DNA replication and histone synthesis is reciprocal.

Oxygen sufficiency controls TOP mRNA translation via the TSC-Rheb-mTOR pathway in a 4E-BP-independent manner

Wed, 06/11/2014 - 11:15am

Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode components of the translational apparatus. This mode of regulation involves TSC and Rheb, as knockout of TSC1 or TSC2 or overexpression of Rheb rescued TOP mRNA translation in oxygen-deprived cells. Stress-induced translational repression of these mRNAs closely correlates with the hypophosphorylated state of 4E-BP, a translational repressor. However, a series of 4E-BP loss- and gain-of-function experiments disprove a cause-and-effect relationship between the phosphorylation status of 4E-BP and the translational repression of TOP mRNAs under oxygen or growth factor deprivation. Furthermore, the repressive effect of anoxia is similar to that attained by the very efficient inhibition of mTOR activity by Torin 1, but much more pronounced than raptor or rictor knockout. Likewise, deficiency of raptor or rictor, even though it mildly downregulated basal translation efficiency of TOP mRNAs, failed to suppress the oxygen-mediated translational activation of TOP mRNAs. Finally, co-knockdown of TIA-1 and TIAR, two RNA-binding proteins previously implicated in translational repression of TOP mRNAs in amino acid-starved cells, failed to relieve TOP mRNA translation under other stress conditions. Thus, the nature of the proximal translational regulator of TOP mRNAs remains elusive. of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.