Identification of tissue transglutaminase protein network

2009/2010Tissue transglutaminase (TG2) is a multifunctional enyzme involved in cell growth and differentiantion, receptor mediated endocytosis, cell adhesion and morphology, stabilization of extracellular matrix, membrane trafficking and structure/function, signal transduction, regulation of cytoskeleton and apoptosis. Multiple lines of evidence suggest an involvement of TG2 autoimmune diseases, cancer and in neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, Huntington's disease and Parkinson's disease. In all of the neurodegenerative diseases examined to date, TG2 activity is upregulated in selectively vulnerable brain regions, TG2 proteins are associated with inclusion bodies characteristic of the diseases, and prominent proteins in the inclusion bodies are modified by TG2 enzyme. It is important to identify TG2 substrates as they may offer an understanding of how the TG2-catalyzed post-translational modification has an impact on physiology and disease. Identification of these substrates may lead to novel drug targets and new diagnostic markers for several TG2-related diseases. A variety of different methods have been proposed for the identification of TG2 substrates. In this work we applied a new method for identification of TG2 substrates (interactors) by using a selection of cDNA phage display libraries followed by massive gene sequencing with 454 system. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential substrates and interactors, which were subsequently confirmed in functional assays. Within the identified clones, some had been previously described as interacting proteins (fibronectin, SMOC1, EIF4G2, MYO18A, GSTO2), while others were new. When compared to standard systems, such as microtiter ELISA, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction. We expect that this approach to library and selection analysis can also be extended to other methods traditionally used to study protein-protein interactions, as well as to the study of the selection of peptides and antibodies by phage display.L'enzima transglutaminasi tissutale è un enzima multifunzionale. Questa proteina gioca un ruolo importante durante lo sviluppo, crescita e differenziamento cellulare, endocitosi mediata da recettore, adesione e morfologia cellulare, stabilizzazione della matrice extracellulare, traffico e struttura/funzione di membrana, trasduzione del segnale, regolazione del citoscheletro ed apoptosi. Molteplici evidenze indicano un coinvolgimento di TG2 in diverse patologie neurodegenerative, incluso il morbo di Alzheimer, la paralisi progressiva supranucleare, il morbo di Huntington e quello di Parkinson. In tutte le malattie neurodegenerative esaminate finora, l'attività della TG2 è aumentata in specifiche regioni cerebrali e le proteine sono associate in corpi d’inclusione caratteristici di tali patologie dove vengono modificate dall'enzima TG2. E’ importante identificare i substrati della TG2 per comprendere come le modifiche post-traduzionali introdotte da questo enzima siano coinvolte nella patogenesi delle suddette malattie. Molteplici metodiche sperimentali sono state proposte ai fini dell'identificazione dei substrati della TG2. In questo lavoro è stato applicato un nuovo metodo per l’identificazione dei substrati della TG2 (interattori), selezionando una libreria di cDNA espressa come phage display, seguito da un sequenziamento genico massivo utilizzando il sistema 454 Life Sciences. La classificazione e l’analisi di più di 120,000 sequenze di DNA ha permesso di identificare molti substrati e potenziali interattori, che sono stati successivamente confermati con le analisi funzionali. All’interno dei cloni identificati, alcuni erano già stati precedentemente descritti come proteine interagenti (interattori) (fibronectina, SMOC1, EIF4G1, MYO18A, GSTO2), mentre altri sono stati identificati come nuovi. Nella comparazione con i metodi standard, come, ad esempio, ELISA, il metodo qui descritto risulta enormemente più rapido e fornisce un numero molto maggiore di informazioni relative alle interazioni analizzate, permettendo quindi una migliore caratterizzazione di sistemi complessi. Ad esempio, nel caso della fibronectina, è stato possibile identificare i domini specifici coinvolti nell’interazione. Prevediamo che questo approccio per l’analisi e la selezione di librerie, possa essere applicato anche ad altri metodi tradizionalmente usati per lo studio di interazioni proteina- proteina, così come allo studio di selezioni di peptidi e anticorpi tramite la tecnica del phage display.XXIII Ciclo197

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction

System-level analyses of keystone genes required for mammalian tooth development

When a null mutation of a gene causes a complete developmental arrest, the gene is typically considered essential for life. Yet, in most cases, null mutations have more subtle effects on the phenotype. Here we used the phenotypic severity of mutations as a tool to examine system-level dynamics of gene expression. We classify genes required for the normal development of the mouse molar into different categories that range from essential to subtle modification of the phenotype. Collectively, we call these the developmental keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show highly elevated expression levels for genes essential for the progression of tooth development, a result reminiscent of essential genes in single-cell organisms. Elevated expression levels of progression genes were also detected in developing rat molars, suggesting evolutionary conservation of this system-level dynamics. Single-cell RNAseq analyses of developing mouse molars reveal that even though the size of the expression domain, measured in the number of cells, is the main driver of organ-level expression, progression genes show high cell-level transcript abundances. Progression genes are also upregulated within their pathways, which themselves are highly expressed. In contrast, a high proportion of the genes required for normal tooth patterning are secreted ligands that are expressed in fewer cells than their receptors and intracellular components. Overall, even though expression patterns of individual genes can be highly different, conserved system-level principles of gene expression can be detected using phenotypically defined gene categories.Peer reviewe

Transcriptomic landscape of early hair follicle and epidermal development

Morphogenesis of ectodermal organs, such as hair, tooth, and mammary gland, starts with the formation of local epithelial thickenings, or placodes, but it remains to be determined how distinct cell types and differ-entiation programs are established during ontogeny. Here, we use bulk and single-cell transcriptomics and pseudotime modeling to address these questions in developing hair follicles and epidermis and pro-duce a comprehensive transcriptomic profile of cellular populations in the hair placode and interplacodal epithelium. We report previously unknown cell populations and marker genes, including early suprabasal and genuine interfollicular basal markers, and propose the identity of suprabasal progenitors. By uncover-ing four different hair placode cell populations organized in three spatially distinct areas, with fine gene expression gradients between them, we posit early biases in cell fate establishment. This work is accom-panied by a readily accessible online tool to stimulate further research on skin appendages and their pro-genitors.Peer reviewe

RhoB is associated with the anti-angiogenic effects of celiac patient transglutaminase 2-targeted autoantibodies

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used

Transglutaminase 2-specific coeliac disease autoantibodies induce morphological changes and signs of inflammation in the small-bowel mucosa of mice

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease

Internkommunikation av ett varumärke - En jämförande studie inom Norrköpings kommun

Norrköpings kommun är en organisation med många verksamhetsområden. Att få alla verksamheter att uppfatta och ta till sig samma värdegrund kan vara svårt, men viktigt om varumärket ska kommuniceras korrekt utåt av alla verksamheter. En medarbetarenkät som gjorts av kommunen ligger till grund för studien då denna visar på medarbetarna inte tänker på sin arbetsplats som en del av kommun. Därför vidtogs åtgärder för att öka kännedomen om varumärket Norrköpings kommun med hjälp av kommunikationsplattformen  Let’s Create Norrköping. Kommunen vet dock ännu inte om medarbetarna har tagit till sig informationen och vad de tycker om varumärket. För att undersöka detta har en fallstudie genomförts som syftar till att undersöka hur varumärket Norrköping kommuniceras genom Let´s Create Norrköping samt hur det uppfattas av medarbetarna på Norrköpings kommun. För att besvara syftet har en datainsamling gjorts vilken utgörs av intervjuer i två delar. I den första delen utfördes en intervju med varumärkesutvecklare Tina Vennerholm. Den gick ut på att klargöra budskapet med Let´s Create Norrköping och bakgrunden till kommunikationsplattformen. Den andra delen genomfördes med fem medarbetare på Norrköpings kommun och gick ut på att undersöka deras uppfattning av varumärket och hur de tar emot information på sina respektive arbetsplatser. De slutsatser som kan dras utifrån studien är att medarbetarna saknar kommunens närvaro på deras respektive arbetsplatser men att de överlag är positivt inställda till kommunikationsplattformen Let´s Create Norrköping. Det framgår dock att de ej fått någon kännedom om Let´s Create Norrköping internt utan att den information de fått kommer från den externa marknadsföring de tar del av i form av invånare i Norrköping. Detta leder till att medarbetarna har svårt att implementera och förstå Let´s Create Norrköpings budskap i sitt arbete. Rekommendationerna som ges till Norrköpings kommun är att synas mer på de olika arbetsplatserna, visa upp vilka från kommunen som ansvariga för kommunikation och liknande. Detta för att medarbetarna ska få en personlig koppling till källan för informationen och på så sätt kunna ta till sig och acceptera den lättare. Medarbetarna har mycket positiva bilder av Norrköping som stad. En rekommendation blir därför att kommunen ska arbeta med att tydligare länka sitt arbete till de olika positiva delar i Norrköpings stad. Detta för att engagera och motivera medarbetarna och få dem att se sin koppling till Norrköpings kommun och det arbete som kommunen utför i staden Norrköping.The municipality of Norrköping is a large organization with lots of different areas of expertise. To make sure that the values are the same throughout the organization is a heavy task but important if you want the brand to be communicated in the right way in all instances of the organization. An internal review of the employees opinions takes place every other year with the last one taking place in 2013. The results from the review made the municipality of Norrköping realise that they needed to increase their internal work with brand knowledge. The municipality of Norrköping took action and created a platform of communications called Let´s Create Norrköping. The problem they stand before now is that they're not sure if the employees have interpreted the information the correct way regarding the brand. This case study aims to investigate how the brand Norrköping is communicated throughout the plattform Let´s Create Norrköping and how the employees perceives the brand of the municipality of Norrköping. To answer the cause of the report a data collection has been made with interviews divided into two parts. The first part consists of an interview with Tina Vennerholm, brand developer at the communications department of the municipality of Norrköping. The main objective of this interviews was to conclude the main objectives of the platform Let´s Create Norrköping. The second part was conducted with five employees within the organization of the municipality of Norrköping. It was conducted to conclude their point of view of the platform Let´s Create Norrköping. Conclusions that can be drawn from the study include that the employees lack the presence of the representatives of the municipality of Norrköping at their working places. The employees have a positive opinion regarding the platform Let´s Create Norrköping although they have not received any internal information regarding the plattform. The reason to their positive opinions ought to be that they have been affected by external marketing regarding the plattform. This leads to the employees having a hard time with implementing the message of the platform in their work. Recommendations given to the municipality of Norrköping is to be visible at the various working places, to show the worker which people who are responsible for the communication at their workplace. to give the employees a personal connection to the source of the information that is given which makes it easier for the worker to accept and implement the information. The employees all have positive connections to the city of Norrköping. That is why the conductors of this study recommend that the municipality of Norrköping should make a connection to the city with the work that is made by the employees on all the different areas in the organization. This is to engage and motivate the employees and make them see and understand the connection between the city and the organization and the part that they play in it

Functionality of vessels in mice in the presence of celiac patient-derived monoclonal autoantibodies (CD Mab) or their relevant controls (non-CD Mab).

<p>(A) Representative images of mouse matrigel implants were treated with CD Mab or non-CD Mab. Hoechst 33342 (Hoechst) was injected intravenously into a tail vein five minutes prior to euthanasia. The removed implants were stained for blood vessels with anti-von Willebrand factor (vWF)-antibody (green). Blue color indicates Hoechst-stained nuclei (DAPI) and scale bar represents 200 µm. (B) Vascular functionality parameters were determined from matrigel implants (left panel) and the ratio of Hoechst- and vWF-positive area was calculated (right panel). (n = 4 animals per group). Bars represent the average value as percentage + SEM. All data was normalized to the basal group (dotted line). * represents P≤0.05 statistical difference.</p

Cellular behavior is altered in the presence of celiac autoantibodies.

<p>(A) Transmission electron micrographs of mouse aorta ring experiments in the presence of monoclonal celiac patient-derived transglutaminase 2-targeted antibody (CD Mab) or its respective control (non-CD Mab). Arrows indicate the extracellular matrix (ECM)-free area in the leading edges of the cells treated with non-CD Mab and the white dotted line shows an area of densely organized ECM around a cell treated with CD Mab. Scale bar represents 6 µm. (B) Representative images taken from mouse matrigel implants treated with CD Mab or non-CD Mab. Nuclei are shown as blue (DAPI) from the border and center of the implants. Scale bar represents 300 µm.</p