Sirtuins: Sir2-related NAD-dependent protein deacetylases

Silent information regulator 2 (Sir2) proteins, or sirtuins, are protein deacetylases dependent on nicotine adenine dinucleotide (NAD) and are found in organisms ranging from bacteria to humans. In eukaryotes, sirtuins regulate transcriptional repression, recombination, the cell-division cycle, microtubule organization, and cellular responses to DNA-damaging agents. Sirtuins have also been implicated in regulating the molecular mechanisms of aging. The Sir2 catalytic domain, which is shared among all sirtuins, consists of two distinct domains that bind NAD and the acetyl-lysine substrate, respectively. In addition to the catalytic domain, eukaryotic sirtuins contain variable amino- and carboxy-terminal extensions that regulate their subcellular localizations and catalytic activity

The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.

The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic alpha-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria

Negative regulation of DAB2IP by Akt and SCFFbw7 pathways

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCFFbw7. DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1δ. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development

Examining one university-based education leadership preparation program, regarding the use of one set of benchmark standards, for the purpose of informing practice

In November of 2006, the North Carolina State Board of Education voted to discontinue the use of the Interstate School Leadership Licensure Consortium (ISLLC) Standards as the guiding and foundational knowledge base for the training of future educational leaders in North Carolina. In December of 2006, a new set of benchmark standards was established in the form of The North Carolina Standards for School Executives. The purpose of this study was to discover the effectiveness in using the ISLLC Standards for equipping education leadership graduates with the skills needed to be successful in their roles and responsibilities as school leaders. The research study was conducted in two phases: interviews and a survey. In total, 127 individuals participated in one of the two phases of research. Participants were graduates from one of three University of North Carolina at Greensboro Educational Leadership and Cultural Foundations (ELC) programs between the years of 1998-2008. The second phase of research was conducted using a web-based survey software. The study yielded a great deal of usable information regarding the effectiveness of an ISLLC-based education in preparing educational leaders for the roles and responsibilities of the workplace. The study also yielded substantial feedback about the effectiveness of the MSA, Ed.S, and Ed.D programs, as a whole, within the ELC Department at UNC-Greensboro

How do principals conceptualize success: are their actions consistent with their definitions?

My research study explored how principals allocated their time, their perceptions of success, and whether their actions were consistent with their definition of success. Findings revealed participants spent time performing three primary behaviors including communicating with school stakeholders, completing managerial practices, and serving as instructional leaders. Additionally, participants conceptualized success as more than performance on state accountability assessments including components such as academic and behavioral growth, appreciation for education and lifelong learning, the belief in future school and societal success, maturing higher-level thinking skills, and an individualized view of success specific to children. Finally, the significant majority of principals carried out actions that were consistent with their opinions of success. Conversely, the participants who did not perform a majority of practices associated with their perceptions of success were influenced by moderating factors, which emphasized the relationship between contextual and individual characteristics and principals' time allotment

SCFβ-TRCP regulates osteoclastogenesis via promoting CYLD ubiquitination

CYLD negatively regulates the NF-κB signaling pathway and osteoclast differentiation largely through antagonizing TNF receptor-associated factor (TRAF)-mediated K63-linkage polyubiquitination in osteoclast precursor cells. CYLD activity is controlled by IκB kinase (IKK), but the molecular mechanism(s) governing CYLD protein stability remains largely undefined. Here, we report that SCFβ-TRCP regulates the ubiquitination and degradation of CYLD, a process dependent on prior phosphorylation of CYLD at Ser432/Ser436 by IKK. Furthermore, depletion of β-TRCP induced CYLD accumulation and TRAF6 deubiquitination in osteoclast precursor cells, leading to suppression of RANKL-induced osteoclast differentiation. Therefore, these data pinpoint the IKK/β-TRCP/CYLD signaling pathway as an important modulator of osteoclastogenesis

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

Bryan S. Der, Ron Jacak, Brian Kuhlman, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, Andrew D. Ellington , Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, George Georgiou, Andrew D. Ellington, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of AmericaAleksandr E. Miklos, Andrew D. Ellington , Applied Research Laboratories, University of Texas at Austin, Austin, Texas, United States of AmericaSergey Lyskov, Jeffrey J. Gray, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of AmericaBrian Kuhlman, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaReengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ~3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.This work was supported by the Defense Advanced Research Projects Agency (HR-0011-10-1-0052 to A.E.) and the Welch Foundation (F-1654 to A.E.), the National Institutes of Health grants GM073960 (B.K.) and R01-GM073151 (J.G. and S.L.), the Rosetta Commons (S.L.), the National Science Foundation graduate research fellowship (2009070950 to B.D.), the UNC Royster Society Pogue fellowship (B.D.), and National Institutes of Health grant T32GM008570 for the UNC Program in Molecular and Cellular Biophysics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Center for Systems and Synthetic BiologyCellular and Molecular BiologyApplied Research LaboratoriesEmail: [email protected]

Outcome of pregnancy after the mustard operation for transposition of the great arteries with intact ventricular septum

AbstractObjectives. This study examined the outcome of pregnancy in patients with transposition of the great arteries and an intact ventricular septum after a Mustard operation.Background. Before the introduction of surgical treatment, most children with transposition of the great arteries died in early infancy. A number of these patients have now reached their reproductive years. There is little information about the effect of pregnancy on cardiovascular status, particularly the ability of the right ventricle to adjust to the hemodynamic changes of pregnancy. The outcome for the offspring and their risk of congenital heart disease are also unknown.Methods. Twenty-three female late survivors after the Mustard operation >15 years of age were reviewed in relation to the occurrence of pregnancy and its outcome. Serial echocardiographic estimates of right ventricular volume during pregnancy were made in three local patients.Results. Nine women had 15 pregnancies. They were asymptomatic before pregnancy and remained free from cardiac symptoms during each pregnancy. Right ventricular volume in the three patients studied increased during pregnancy but returned to normal at 8 to 11 weeks postpartum. There were 12 live births, 2 spontaneous abortions and 1 intrauterine death. None of the liveborn infants had evidence of congenital heart disease.Conclusions. In this small group of women with good quality late survival after a Mustard operation, pregnancy was well tolerated. We suspect that the incidence of congenital heart disease in infants of mothers with transposition of the great arteries will be at the lower end of the range for mothers with different types of congenital heart disease, but further data will be needed to confirm this

Measuring M2 values for on-wafer vertical cavity surface emitting lasers

We report on M2 measurements taken for on-wafer vertical cavity surface emitting lasers (VCSELs). We measured M2 for oxide-confined VCSELs and photonic crystal (PhC) VCSELs of similar lasing aperture sizes

Characterization of Single-Mode Vertical Cavity Surface-Emitting Lasers

A high-quality single-mode beam is desirable for the efficient use of lasers as light sources for optical data communications and interconnects, however there is little data which characterizes operating ranges and near-field beam qualities of Vertical Cavity Surface Emitting Lasers (VCSELs), which has resulted in a lack of analysis of these devices. Measures of beam quality include beam-quality factor (M2 ), Side-Mode-Suppression-Ratio (SMSR) and RMS linewidth. M2 is a measurement of how closely the beam is to an ideal Gaussian. SMSR is the difference, in dB, between the amplitude of the primary peak and the amplitude of the next highest peak of the output spectrum, with single-mode operation defined by a SMSR \u3e 30 dB. RMS linewidth is a second moment calculation involving the power spectral density, where smaller RMS linewidth indicates higher beam quality. Utilizing a novel vertical M2 setup in which on-wafer VCSEL M2 can be measured, a study was conducted on the relation between M2 , SMSR and RMS linewidth, for various oxide-confined VCSELs of varying aperture sizes and Photonic Crystal (PhC) VCSELs of varying aperture sizes and photonic crystal configurations. First, the operating range of the VCSEL was determined utilizing a Semiconductor Parameter Analyzer to obtain the LIV characteristics. Along with this measurement, spectral data was collected using an Optical Spectrum Analyzer at several key operating points, which allowed the RMS linewidths and SMSRs of the devices to be calculated at these points. The novel beam-profiler setup was used to measure the device’s M2 . Initial results show a strong correlation between the measures of beam quality, with increasing SMSR, corresponding to M2 values closer to 1, and single-mode operation characterized by a M2 of less than 1.5. A strong correlation between RMS linewidth and M2 was also seen, with increasing RMS linewidths corresponding to an increase in M2