Funktionelle Analyse der onkologisch relevanten Protease Threonin-Aspartase 1

Krebs stellt eine der häufigsten Todesursachen in Europa dar. Grundlage für eine langfristige Verbesserung des Behandlungserfolgs ist ein molekulares Verständnis der Mechanismen, welche zur Krankheitsentstehung beitragen. In diesem Zusammenhang spielen Proteasen nicht nur eine wichtige Rolle, sondern stellen auch bei vielerlei Erkrankungen bereits anerkannte Zielstrukturen derzeitiger Behandlungsstrategien dar. Die Protease Threonin Aspartase 1 (Taspase1) spielt eine entscheidende Rolle bei der Aktivierung von Mixed Lineage Leukemia (MLL)-Fusionsproteinen und somit bei der Entstehung aggressiver Leukämien. Aktuelle Arbeiten unterstreichen zudem die onkologische Relevanz von Taspase1 auch für solide Tumore. Die Kenntnisse über die molekularen Mechanismen und Signalnetzwerke, welche für die (patho)biologischen Funktionen von Taspase1 verantwortlich sind, stellen sich allerdings noch immer als bruchstückhaft dar. Um diese bestehenden Wissenslücken zu schließen, sollten im Rahmen der Arbeit neue Strategien zur Inhibition von Taspase1 erarbeitet und bewertet werden. Zusätzlich sollten neue Einsichten in evolutionären Funktionsmechanismen sowie eine weitergehende Feinregulation von Taspase1 erlangt werden. Zum einen erlaubte die Etablierung und Anwendung eines zellbasierten Taspase1-Testsystem, chemische Verbindungen auf deren inhibitorische Aktivität zu testen. Überraschenderweise belegten solch zelluläre Analysen in Kombination mit in silico-Modellierungen eindeutig, dass ein in der Literatur postulierter Inhibitor in lebenden Tumorzellen keine spezifische Wirksamkeit gegenüber Taspase1 zeigte. Als mögliche Alternative wurden darüber hinaus Ansätze zur genetischen Inhibition evaluiert. Obwohl publizierte Studien Taspase1 als ααββ-Heterodimer beschreiben, konnte durch Überexpression katalytisch inaktiver Mutanten kein trans-dominant negativer Effekt und damit auch keine Inhibition des wildtypischen Enzyms beobachtet werden. Weiterführende zellbiologische und biochemische Analysen belegten erstmalig, dass Taspase1 in lebenden Zellen in der Tat hauptsächlich als Monomer und nicht als Dimer vorliegt. Die Identifizierung evolutionär konservierter bzw. divergenter Funktionsmechanismen lieferte bereits in der Vergangenheit wichtige Hinweise zur Inhibition verschiedenster krebsrelevanter Proteine. Da in Drosophila melanogaster die Existenz und funktionelle Konservierung eines Taspase1-Homologs postuliert wurde, wurde in einem weiteren Teil der vorliegenden Arbeit die evolutionäre Entwicklung der Drosophila Taspase1 (dTaspase1) untersucht. Obwohl Taspase1 als eine evolutionär stark konservierte Protease gilt, konnten wichtige Unterschiede zwischen beiden Orthologen festgestellt werden. Neben einem konservierten autokatalytischen Aktivierungsmechanismus besitzt dTaspase1 verglichen mit dem humanen Enzym eine flexiblere Substraterkennungs-sequenz, was zu einer Vergrößerung des Drosophila-spezifischen Degradoms führt. Diese Ergebnisse zeigen des Weiteren, dass zur Definition und Vorhersage des Degradoms nicht nur proteomische sondern auch zellbiologische und bioinformatische Untersuchungen geeignet und notwendig sind. Interessanterweise ist die differentielle Regulation der dTaspase1-Aktivität zudem auf eine veränderte intrazelluläre Lokalisation zurückzuführen. Das Fehlen von in Vertebraten hochkonservierten aktiven Kernimport- und nukleolären Lokalisationssignalen erklärt, weshalb dTaspase1 weniger effizient nukleäre Substrate prozessiert. Somit scheint die für die humane Taspase1 beschriebene Regulation von Lokalisation und Aktivität über eine Importin-α/NPM1-Achse erst im Laufe der Entwicklung der Vertebraten entstanden zu sein. Es konnte also ein bislang unbekanntes evolutionäres Prinzip identifiziert werden, über welches eine Protease einen Transport- bzw. Lokalisations-basierten Mechanismus zur Feinregulation ihrer Aktivität „von der Fliege zum Menschen“ nutzt. Eine weitere Möglichkeit zur dynamischen Funktionsmodulation bieten post-translationale Modifikationen (PTMs) der Proteinsequenz, zu welcher Phosphorylierung und Acetylierung zählen. Interessanterweise konnte für die humane Taspase1 über den Einsatz unabhängiger Methoden einschließlich massenspektrometrischer Analysen eine Acetylierung durch verschiedene Histon-Acetyltransferasen (HATs) nachgewiesen werden. Diese Modifikation erfolgt reversibel, wobei vor allem die Histon-Deacetylase HDAC1 durch Interaktion mit Taspase1 die Deacetylierung der Protease katalysiert. Während Taspase1 in ihrer aktiven Konformation acetyliert vorliegt, kommt es nach Deacetylierung zu einer Reduktion ihrer enzymatischen Aktivität. Somit scheint die Modulation der Taspase1-Aktivität nicht allein über intra-proteolytische Autoaktivierung, Transport- und Interaktionsmechanismen, sondern zudem durch post-translationale Modifikationen gesteuert zu werden. Zusammenfassend konnten im Rahmen dieser Arbeit entscheidende neue Einblicke in die (patho)biologische Funktion und Feinregulation der Taspase1 gewonnen werden. Diese Ergebnisse stellen nicht nur einen wichtigen Schritt in Richtung eines verbesserten Verständnis der „Taspase1-Biologie“, sondern auch zur erfolgreichen Inhibition und Bewertung der krebsrelevanten Funktion dieser Protease dar.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc.

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.

The SLC3 and SLC7 families of amino acid transporters

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Cell survival in early embryogenesis requires proper dosage of GCN5 and p300

Histone acetylation is a central event in transcriptional activation. The importance of this modification in mammalian development is highlighted by knockout studies that revealed loss of the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality. Furthermore, early embryogenesis is sensitive to the dosage of p300 and CBP since double p300 +/−CBP+/− heterozygotes die in utero, although either single heterozygote survives. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of HATs interact functionally in vivo, we created mice lacking one or more allele of p300, GCN5 or PCAF. As expected, we found that mice heterozygous for any one of these null alleles are viable. The majority of GCN5 p300 double heterozygotes also survive to adulthood with no apparent abnormalities. However, a portion of these mice die prior to birth. These embryos are developmentally stunted and exhibit increased apoptosis compared to wild type or single GCN5 or p300 heterozygous littermates at E8.5. Tissue specification is unaffected in these embryos but organ formation is compromised. In contrast, no abnormalities were observed in mice harboring mutations in both PCAF and p300 , emphasizing the specificity of HAT functions in mammalian development. ^ Since GCN5 null embryos die early in embryogenesis because of a marked increase in apoptosis, studies of its function and mechanism in late development and in tissue specific differentiation are precluded. Here, we also report the establishment of a GCN5 null embryonic stem cell line and a conditional floxGCN5 mouse line, which will serve as powerful genetic tools to examine in depth the function of GCN5 in mammalian development and in adult tissues. ^

Diseño de una interfaz de representación gráfica de la base de datos de HRTF

El actual proyecto consiste en la creación de una interfaz gráfica de usuario (GUI) en entorno de MATLAB que realice una representación gráfica de la base de datos de HRTF (Head-Related Transfer Function). La función de transferencia de la cabeza es una herramienta muy útil en el estudio de la capacidad del ser humano para percibir su entorno sonoro, además de la habilidad de éste en la localización de fuentes sonoras en el espacio que le rodea. La HRTF biaural (terminología para referirse al conjunto de HRTF del oído izquierdo y del oído derecho) en sí misma, posee información de especial interés ya que las diferencias entre las HRTF de cada oído, conceden la información que nuestro sistema de audición utiliza en la percepción del campo sonoro. Por ello, la funcionalidad de la interfaz gráfica creada presenta gran provecho dentro del estudio de este campo. Las diferencias interaurales se caracterizan en amplitud y en tiempo, variando en función de la frecuencia. Mediante la transformada inversa de Fourier de la señal HRTF, se obtiene la repuesta al impulso de la cabeza, es decir, la HRIR (Head-Related Impulse Response). La cual, además de tener una gran utilidad en la creación de software o dispositivos de generación de sonido envolvente, se utiliza para obtener las diferencias ITD (Interaural Time Difference) e ILD (Interaural Time Difference), comúnmente denominados “parámetros de localización espacial”. La base de datos de HRTF contiene la información biaural de diferentes puntos de ubicación de la fuente sonora, formando una red de coordenadas esféricas que envuelve la cabeza del sujeto. Dicha red, según las medidas realizadas en la cámara anecoica de la EUITT (Escuela Universitaria de Ingeniería Técnica de Telecomunicación), presenta una precisión en elevación de 10º y en azimut de 5º. Los receptores son dos micrófonos alojados en el maniquí acústico llamado HATS (Hats and Torso Simulator) modelo 4100D de Brüel&Kjaer. Éste posee las características físicas que influyen en la percepción del entorno como son las formas del pabellón auditivo (pinna), de la cabeza, del cuello y del torso humano. Será necesario realizar los cálculos de interpolación para todos aquellos puntos no contenidos en la base de datos HRTF, este proceso es sumamente importante no solo para potenciar la capacidad de la misma sino por su utilidad para la comparación entre otras bases de datos existentes en el estudio de este ámbito. La interfaz gráfica de usuario está concebida para un manejo sencillo, claro y predecible, a la vez que interactivo. Desde el primer boceto del programa se ha tenido clara su filosofía, impuesta por las necesidades de un usuario que busca una herramienta práctica y de manejo intuitivo. Su diseño de una sola ventana reúne tanto los componentes de obtención de datos como los que hacen posible la representación gráfica de las HRTF, las HRIR y los parámetros de localización espacial, ITD e ILD. El usuario podrá ir alternando las representaciones gráficas a la vez que introduce las coordenadas de los puntos que desea visualizar, definidas por phi (elevación) y theta (azimut). Esta faceta de la interfaz es la que le otorga una gran facilidad de acceso y lectura de la información representada en ella. Además, el usuario puede introducir valores incluidos en la base de datos o valores intermedios a estos, de esta manera, se indica a la interfaz la necesidad de realizar la interpolación de los mismos. El método de interpolación escogido es el de la ponderación de la distancia inversa entre puntos. Dependiendo de los valores introducidos por el usuario se realizará una interpolación de dos o cuatro puntos, siendo éstos limítrofes al valor introducido, ya sea de phi o theta. Para añadir versatilidad a la interfaz gráfica de usuario, se ha añadido la opción de generar archivos de salida en forma de imagen de las gráficas representadas, de tal forma que el usuario pueda extraer los datos que le interese para cualquier valor de phi y theta. Se completa el presente proyecto fin de carrera con un trabajo de investigación y estudio comparativo de la función y la aplicación de las bases de datos de HRTF dentro del marco científico y de investigación. Esto ha hecho posible concentrar información relacionada a través de revistas científicas de investigación como la JAES (Journal of the Audio Engineering Society) o la ASA (Acoustical Society of America), además, del IEEE ( Institute of Electrical and Electronics Engineers) o la “Web of knowledge” entre otras. Además de realizar la búsqueda en estas fuentes, se ha optado por vías de información más comunes como Google Académico o el portal de acceso “Ingenio” a los todos los recursos electrónicos contenidos en la base de datos de la universidad. El estudio genera una ampliación en el conocimiento de la labor práctica de las HRTF. La mayoría de los estudios enfocan sus esfuerzos en mejorar la percepción del evento sonoro mediante su simulación en la escucha estéreo o multicanal. A partir de las HRTF, esto es posible mediante el análisis y el cálculo de datos como pueden ser las regresiones, siendo éstas muy útiles en la predicción de una medida basándose en la información de la actual. Otro campo de especial interés es el de la generación de sonido 3D. Mediante la base de datos HRTF es posible la simulación de una señal biaural. Se han diseñado algoritmos que son implementados en dispositivos DSP, de tal manera que por medio de retardos interaurales y de diferencias espectrales es posible llegar a un resultado óptimo de sonido envolvente, sin olvidar la importancia de los efectos de reverberación para conseguir un efecto creíble de sonido envolvente. Debido a la complejidad computacional que esto requiere, gran parte de los estudios coinciden en desarrollar sistemas más eficientes, llegando a objetivos tales como la generación de sonido 3D en tiempo real. ABSTRACT. This project involves the creation of a Graphic User Interface (GUI) in the Matlab environment which creates a graphic representation of the HRTF (Head-Related Transfer Function) database. The head transfer function is a very useful tool in the study of the capacity of human beings to perceive their sound environment, as well as their ability to localise sound sources in the area surrounding them. The binaural HRTF (terminology which refers to the HRTF group of the left and right ear) in itself possesses information of special interest seeing that the differences between the HRTF of each ear admits the information that our system of hearing uses in the perception of each sound field. For this reason, the functionality of the graphic interface created presents great benefits within the study of this field. The interaural differences are characterised in space and in time, varying depending on the frequency. By means of Fourier's transformed inverse of the HRTF signal, the response to the head impulse is obtained, in other words, the HRIR (Head-Related Impulse Response). This, as well as having a great use in the creation of software or surround sound generating devices, is used to obtain ITD differences (Interaural Time Difference) and ILD (Interaural Time Difference), commonly named “spatial localisation parameters”. The HRTF database contains the binaural information of different points of sound source location, forming a network of spherical coordinates which surround the subject's head. This network, according to the measures carried out in the anechoic chamber at the EUITT (School of Telecommunications Engineering) gives a precision in elevation of 10º and in azimuth of 5º. The receivers are two microphones placed on the acoustic mannequin called HATS (Hats and Torso Simulator) Brüel&Kjaer model 4100D. This has the physical characteristics which affect the perception of the surroundings which are the forms of the auricle (pinna), the head, neck and human torso. It will be necessary to make interpolation calculations for all those points which are not contained the HRTF database. This process is extremely important not only to strengthen the database's capacity but also for its usefulness in making comparisons with other databases that exist in the study of this field. The graphic user interface is conceived for a simple, clear and predictable use which is also interactive. Since the first outline of the program, its philosophy has been clear, based on the needs of a user who requires a practical tool with an intuitive use. Its design with only one window unites not only the components which obtain data but also those which make the graphic representation of the HRTFs possible, the hrir and the ITD and ILD spatial location parameters. The user will be able to alternate the graphic representations at the same time as entering the point coordinates that they wish to display, defined by phi (elevation) and theta (azimuth). The facet of the interface is what provides the great ease of access and reading of the information displayed on it. In addition, the user can enter values included in the database or values which are intermediate to these. It is, likewise, indicated to the interface the need to carry out the interpolation of these values. The interpolation method is the deliberation of the inverse distance between points. Depending on the values entered by the user, an interpolation of two or four points will be carried out, with these being adjacent to the entered value, whether that is phi or theta. To add versatility to the graphic user interface, the option of generating output files in the form of an image of the graphics displayed has been added. This is so that the user may extract the information that interests them for any phi and theta value. This final project is completed with a research and comparative study essay on the function and application of HRTF databases within the scientific and research framework. It has been possible to collate related information by means of scientific research magazines such as the JAES (Journal of the Audio Engineering Society), the ASA (Acoustical Society of America) as well as the IEEE (Institute of Electrical and Electronics Engineers) and the “Web of knowledge” amongst others. In addition to carrying out research with these sources, I also opted to use more common sources of information such as Academic Google and the “Ingenio” point of entry to all the electronic resources contained on the university databases. The study generates an expansion in the knowledge of the practical work of the HRTF. The majority of studies focus their efforts on improving the perception of the sound event by means of its simulation in stereo or multichannel listening. With the HRTFs, this is possible by means of analysis and calculation of data as can be the regressions. These are very useful in the prediction of a measure being based on the current information. Another field of special interest is that of the generation of 3D sound. Through HRTF databases it is possible to simulate the binaural signal. Algorithms have been designed which are implemented in DSP devices, in such a way that by means of interaural delays and wavelength differences it is possible to achieve an excellent result of surround sound, without forgetting the importance of the effects of reverberation to achieve a believable effect of surround sound. Due to the computational complexity that this requires, a great many studies agree on the development of more efficient systems which achieve objectives such as the generation of 3D sound in real time.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

Epstein–Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter

The Epstein–Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs). Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF. P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography. EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300. In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression. Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect. In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity. Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.

Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

In-House Risk

Over the last thirty years or so, as the number of in-house counsel rose and their role increased in scope and prominence, increased attention has been given the various challenges these lawyers face under the ABA Model Rules of Professional Conduct, from figuring out who is the client the in-house lawyer represents, to navigating conflicts of interest, maintaining independence, and engaging in a multijurisdictional practice of law. Less attention, to date, has been given to business risk assessment, perhaps in part because that function appears to be part of in-house counsel’s role as a business person rather than as a lawyer. Overlooking the role of in-house counsel in assessing risk, however, is a risky proposition, because risk assessment constitutes for some in-house counsel a significant aspect of their role, a role that in turn informs and shapes how in-house counsel perform other more overtly legal tasks. For example, wearing her hat as General Counsel, a lawyer for the entity-client may opine and explain issues of compliance with the law. Wearing her hat as the Chief Legal Officer, however, the same lawyer may now be called upon as a member of business management to participate in the decision whether to comply with the law. After outlining some of the traditional challenges faced by in-house counsel under the Rules, this short essay explores risk assessment by in-house counsel and its impact on their role and function under the Rules. It argues that the key to in-house lawyers’ successful navigation of multiple roles, and, in particular, to their effective assessment of business risk is keen awareness of the various hats they are called upon to wear. Navigating these various roles may not be easy for lawyers, whose training and habits of mind often teach them to zoom in on legal risks to the exclusion of business risks. Indeed, law schools continue to teach law students “to think like a lawyer” and law firms, the historical breeding grounds for in-house counsel positions, in a world of increased specialization master the narrower contemplation of legal questions. Yet the present and future of in-house counsel practice demand of its practitioners the careful and gradual coming to terms, buildup and mastery of business risk analysis skills, alongside the cultivation of traditional legal risk analysis tools.

Journal of College Disorders, 1788-1797

This journal contains entries about various student "disorders" which occurred during Eliphalet Pearson’s tenure at Harvard. Daily entries describe a wide range of students’ rebellious conduct, which included: hissing at speakers in chapel, throwing snowballs and stones at College buildings and people (including tutors and then-President Joseph Willard), disrupting lectures by scraping chairs and feet, breaking windows, intoxication, moving and breaking furniture, stealing firewood, firing pistols, building bonfires, stealing supplies (food, cider and candles), throwing food and utensils during meals, stealing Bibles, wearing hats indoors, filling door locks with stones, drawing on lecture room walls with gravel, and silencing the morning chapel bell by filling it with molten pewter plates (stolen from the kitchen). There are also entries pertaining to more malicious offenses, including the drowning of a dog in a well. Several entries describe meetings of the College government to determine the appropriate punishments for each offense. Students were often fined, expelled, or suspended ("rusticated") for their unruly behavior.

Lyndon, Saline and Ann Arbor (Mich.) residences and business, ca. 1874

Left to right: Woodside Res. of N.B. Cole, Ann Arbor, Mich. for sale; Res. of John K. Yocum, Sec. 30, Lyndon Tp. Mich; Corner Main & Liberty Sts., Ann Arbor, Mich.; C. Parsons, dealer in dry goods, boots & shoes, hats, caps &c., Saline Mich. Publication information: Chicago, Ill. : Everts & Stewart, 1874.

Swing Out

Daybook, image #28