The Mechanism of Action of SOCS2 and its Role in Metabolism and Growth

A well-known function of Growth Hormone (GH) is the regulation of postnatal longitudinal growth but it also affects other biological processes, for instance metabolism and inflammation. Actions of GH are tightly regulated at several levels and by several different factors and are initiated by GH binding to membrane bound GH receptors (GHR). The intracellular signaling of GH and other related hormones and cytokines is predominately mediated by the JAK-STAT pathway. This pathway is regulated in a negative feedback manner by the Suppressors of Cytokine Signaling (SOCS) family of proteins. One of the family members, SOCS2, is intimately tied to GH by virtue of the phenotype that results from its absence. SOCS2-/- mice are 40% larger than wildtype littermates due to increased GH sensitivity. Here, the molecular mechanism behind SOCS2s negative regulation of GH signaling, and its effects on metabolism and inflammation are described. We demonstrate that SOCS2 assembles a canonical E3 ubiquitin ligase complex with Elongin B, Elongin C, Cullin 5 and Rbx2 and that this complex has intrinsic E3 ligase activity in vitro. Overexpression of SOCS2 and its complex members leads to ubiquitination and proteasomal degradation of the GHR. We also outline the importance of the different domains of SOCS2, and demonstrate the necessity of the SOCS-box for proper SOCS2 activity. In a follow up study the claim that the naturally occurring Ser52Asn polymorphism of SOCS2 affects its activity and may contribute to acromegaly in humans was investigated. The Ser52Asn mutant was however found to be as efficient at regulating GH signaling as the wildtype and we conclude that it is unlikely to contribute to increased GH sensitivity. In Paper III the phenotype of SOCS2-/- mice under conditions of dietary stress is described. We report that SOCS2 deletion protects against high fat diet (HFD) induced hepatic steatosis but simultaneously leads to decreased insulin sensitivity. SOCS2-/- mice were found to have increased triglyceride output from the liver but also increased plasma levels of proinflammatory cytokines without apparent macrophage infiltration. In vitro examination of macrophages revealed increased phagocytic activity and cytokine production in the absence of SOCS2 and suggests a direct role for SOCS2 in the regulation of TLR4 signaling. Finally, the results of a screening effort to identify SOCS2-modulating, drug-like molecules are included. We have identified a prospective hit that binds to and inhibits SOCS2 activity in vitro. In summary, SOCS forms an E3 ligase complex which targets the GHR for degradation. This forms the molecular basis of its physiological actions. SOCS2-/- mice are protected from HFD induced hepatic steatosis but suffer from deteriorated insulin sensitivity related to increased inflammation

Proteogenomics refines the molecular classification of chronic lymphocytic leukemia

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling

TcellSubC: An Atlas of the Subcellular Proteome of Human T Cells

We have curated an in-depth subcellular proteomic map of primary human CD4+ T cells, divided into cytosolic, nuclear and membrane fractions generated by an optimized fractionation and HiRIEF-LC-MS/MS workflow for limited amounts of primary cells. The subcellular proteome of T cells was mapped under steady state conditions, as well as upon 15 min and 1 h of T cell receptor (TCR) stimulation, respectively. We quantified the subcellular distribution of 6,572 proteins and identified a subset of 237 potentially translocating proteins, including both well-known examples and novel ones. Microscopic validation confirmed the localization of selected proteins with previously known and unknown localization, respectively. We further provide the data in an easy-to-use web platform to facilitate re-use, as the data can be relevant for basic research as well as for clinical exploitation of T cells as therapeutic targets

SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/fl Actin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21−/− mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.publishedVersio

Proteogenomic analysis of acute myeloid leukemia associates relapsed disease with reprogrammed energy metabolism both in adults and children

Despite improvement of current treatment strategies and novel targeted drugs, relapse and treatment resistance largely determine the outcome for acute myeloid leukemia (AML) patients. To identify the underlying molecular characteristics, numerous studies have been aimed to decipher the genomic- and transcriptomic landscape of AML. Nevertheless, further molecular changes allowing malignant cells to escape treatment remain to be elucidated. Mass spectrometry is a powerful tool enabling detailed insights into proteomic changes that could explain AML relapse and resistance. Here, we investigated AML samples from 47 adult and 22 pediatric patients at serial time-points during disease progression using mass spectrometry-based in-depth proteomics. We show that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse. Further, higher levels of granzymes and lower levels of the anti-inflammatory protein CR1/CD35 suggest an inflammatory signature promoting disease progression. Finally, through a proteogenomic approach, we detected novel peptides, which present a promising repertoire in the search for biomarkers and tumor-specific druggable targets. Altogether, this study highlights the importance of proteomic studies in holistic approaches to improve treatment and survival of AML patients.Peer reviewe

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia.

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET

District heating system analysis within the urban transformation of Kiruna

The urban transformation that is taking place in Kiruna, a Swedish town well above the Arctic Circle in the sub-arctic climate, is due to the ground deformation caused by iron ore mining and it is affecting all the infrastructures of the town. This thesis focuses on the studies about the district heating for the town of Kiruna. In the field of district heating it is important to have a good knowledge about network behavior, especially if network structure is meshed, in order to understand how the flow is distributed in each pipe within the network. This thesis describes the development of a method for the simulation of district heating networks that makes it possible to analyze and study complex networks with meshed structure, something that has not been possible before. The thermal losses for each pipe type are required as a fundamental input for the simulation. A fictitious pipe series has been created so that its loss matches the losses in the real network. It has been found that the created series is close to the series with the highest losses that is manufactured today. When redesigning the network structure to deal with the urban transformation, an investigation is performed regarding how new low energy building can be heated. This investigation shows that the only heating system that fulfills the guidelines stated by the Swedish authorities about indoor climate is the floor heating. A techno-economical evaluation shows that the cheapest heating technology is an air to air heat pump. In order to make district heating attractive as a heating source for house owners a vigorous price reduction is needed.Godkänd; 2014; 20141002 (matves); Nedanstående person kommer att hålla licentiatseminarium för avläggande av teknologie licentiatexamen. Namn: Mattias Vesterlund Ämne: Energiteknik/Energy Engineering Uppsats: District Heating System Analysis within the Urban Transformation of Kiruna Examinator: Professor Jan Dahl, Institutionen för teknikvetenskap och matematik, Luleå tekniska universitet Diskutant: Magnus Johansson, Luleå Energi Tid: Torsdag den 27 november 2014 kl 10.00 Plats: E632, Luleå tekniska universite