Studies on the mechanisms of action and physiological relevance of SOCS proteins

Understanding systemic biological pathways and the key cellular mechanisms that dictate disease states, drug response, and altered cellular function in metabolic disorders is a significant challenge. Research in the last 20 years have made it clear that tissue communication, through the actions of endocrine, paracrine or autocrine factors play a significant role in pathogenesis of complex multiorgan diseases such as the metabolic syndrome. The actions of these factors are governed both at the site of production and through mechanisms that regulate the sensitivity of target tissues. The Suppressors of Cytokine Signaling (SOCS) proteins act as negative regulators of the main cytokine and growth factor signaling pathways in multiple tissues and as such have important physiological functions. The molecular basis for SOCS actions as well as their role in the pathogenesis of metabolic diseases is just starting to be understood. All SOCS proteins (SOCS1 to 7 and CIS) are characterized by the presence of structural motifs called SOCS box and a SH2 domain. SOCS are thought to act as substrate recognition subunits of multimeric Cullin/RING E3 ubiquitin ligase complexes. It has been proposed that the SH2 domain mediates the recognition of tyrosine phosphorylated signaling proteins to be targeted for ubiquitination and subsequently, proteasomal degradation, thereby inhibiting cytokine signaling. In this thesis we investigated the mechanisms of action of SOCS2 and SOCS6 in the inhibition of cellular signaling and the physiological consequence of their actions. We demonstrated that both SOCS2 and SOCS6 assemble a canonical ECS (Elongin/Cullin/SOCS) complex through the interaction of SOCS box with Elongin B and C, cullin 5 and Rbx2. We also demonstrated that SOCS2 and SOCS6 exert E3- ligase activity towards the growth hormone (GH) receptor and cKIT proteins, respectively. Our structural and binding studies confirmed the existence of substrate binding motifs mainly in the SH2 domains and the N-terminal domain of both SOCSs. The C-terminus harbours the cullin 5 recognition domain that controls both E3 ligase activity of the complex as well as the SOCS stability. We proposed that extended target recognition domain in the SOCS proteins may serve to broaden their specificity toward various targets and hence their ability to regulate various signaling pathways. On the other hand, the existence of a degradation signal within the Elongin C interacting domain of SOCS proteins may serve to secure their timely actions avoiding competition from SOCS that are not engaged in active E3 ubiquitin ligase complexes. The patho-physiological role of SOCS2 was studied in SOCS2 knockout (SOCS2-/-) mice. In line with the in vitro studies (paper I), we observed an increased GH sensitivity in SOCS2-/- mice, demonstrated by low plasma GH/IGF1 ratio. In the liver, this enhanced sensitivity was manifested through increased VLDL secretion and reduced hepatic triglycerides levels. SOCS2-/- showed reduced hepatic steatosis upon high fat feeding as compared to wild type littermates but also exhibited increased adiposity and fat deposition in the skeletal muscles accompanied by profound systemic insulin resistance. We also demonstrated the involvement of SOCS2 in the regulation of inflammatory pathways. SOCS2-/- mice showed an exacerbated response to a high fat diet, with increased expression of inflammatory cytokines such as IL-6, RANTES, IL1β both in the liver and adipose tissue. We also identified possible mechanisms to explain these phenomena by demonstrating that macrophages isolated from SOCS2-/- mice showed higher phagocytic activity and higher LPS-induced NF-κB activity; indicative of SOCS2 negative regulation of TLR4 signals. Given the short half-life of SOCS proteins in the cells, the regulation of SOCS gene transcription is an important mechanism to control their function. The nuclear receptor LXR has regulatory functions on hepatic lipid metabolism that overlap with those controlled by the GH. Therefore, in order to understand the molecular basis for possible crosstalks between these two pathways, we studies how LXR ligands regulate the GH receptor signaling in liver. We showed that LXR agonist downregulates STAT5b protein levels and suppresses GH receptor activity in hepatocytes through a mechanism involving SREBP1. The regulation by SREBP1 occurs through the modulation of STAT5b protein stability and results in reduced expression of GH target genes such as SOCS2. These results provide a plausible explanation for the hepatosteatosis observed upon LXR agonist treatment in vivo. In conclusion, through structural, in vivo and in vitro studies, we provide mechanistic and functional data on SOCS2 and SOCS6; information that may lead to a better understanding of the distinct physiological functions of these proteins. Given our demonstration on the key role of ubiquitination on SOCS functions, future mechanistic studies of SOCS2 and SOCS6 function should focus on the identification of ubiquitination targets of these proteins. Are the physiological functions ascribed to these proteins a result of the degradation of a few target proteins or do they have many targets? If the later is the case, how is SOCS target-specificity structurally determined and how is their activity regulated in time and cellular location? The demonstration that SOCS2 regulate both GH receptor and TLR4 signaling offers system where these questions can be addressed. At physiological levels, the SOCS2-/- mice constitute a novel model system for the study of the metabolic syndrome with unique features that are relevant to the human disease. In the SOCS2-/- mice, insulin resistance and production of inflammatory cytokine are exacerbated by high fat feeding and associated with obesity and deposition of triacylglycerides in the muscle. In these conditions reduced accumulation of TG in liver is observed. This model presents opportunities for future investigations aiming to distinguish between steatotic versus inflammatory causes for insulin resistance

Frequencies of the Arg16Gly, Gln27Glu and Thr164Ile Adrenoceptor β2 Polymorphisms among Omanis

Objectives: This study aimed to assess the distribution of missense mutations in the adrenoceptor β2 (ADRB2) gene in an Omani cohort. Methods: This study was carried out between May 2014 and March 2015 at the Sultan Qaboos University, Muscat, Oman. Blood samples were taken from 316 unrelated Omani subjects. Genotyping for rs1042713 (c.46A>G, p.Arg16Gly), rs1042714 (c.79C>G, p.Gln27Glu) and rs1800888 (c.491C>T, p.Thr164Ile) polymorphisms was performed by real-time polymerase chain reaction using single nucleotide polymorphism (SNP) genotyping assays. The allelic frequencies of these polymorphisms were estimated on the basis of the observed numbers of specific alleles from the genotype data for male and female subjects. The genotype frequencies for each polymorphism were tested for deviation from the Hardy-Weinberg equilibrium. Results: Gly16 and Glu27 were the most frequent variants found among the cohort (63% and 75%, respectively). The Ile164 variant was not detected in the study population. There was a significant linkage disequilibrium between the rs1042713 and rs1042714 SNPs (r2 = 0.209; P ≤0.001). The most observed haplotypes were Gly16-Gln27 and Arg16-Gln27 (0.37 and 0.38, respectively). The frequency of Gly16-Glu27 was 0.25, comprising all Glu27 carriers. Conclusion: The allelic distribution of variants in this Omani cohort was similar to distributions reported among Caucasian populations

Reference Ranges of Serum Anti-Müllerian Hormone in Healthy Reproductive-aged Omani Women

Objectives: Anti-Müllerian hormone (AMH), a glycoprotein that belongs to the transforming growth factor-beta superfamily, is important for women’s health. We aimed to determine the age-specific reference range of serum AMH in healthy Omani women from reproductive ages to menopause. Methods: This cross-sectional cohort study was conducted among a group of healthy 20–50 years old Omani women. The participants were required to have body mass index < 32 kg/m2 regular periods, no history of chronic illness, polycystic ovary syndrome, or gynecological operation. They were also required to not be using any hormonal contraceptive. Serum concentrations of AMH, follicle-stimulating hormone, luteinizing hormone, progesterone, and hemoglobin A1c were measured. AMH-age nomogram and AMH levels were compared between the six selected age groups. Results: The subjects were 319 Omani women aged 20–50 years. Serum AMH concentrations were found to decrease progressively with increasing age. An exponential model defined as √AMH = 479.02 × 0.91age was selected to explain the reduction in AMH with age (R2 = 0.298). The median AMH levels were 26.61 pmol/L for those aged 20–25 years, 20.89 pmol/L for 26–30 years, 19.92 pmol/L for 31–35 years, 13.71 pmol/L for 36–40 years, 9.24 pmol/L for 41–45 years, and 0.68 pmol/L for 46–50 years. The recommended 2.5thto 97.5thpercentiles of AMH level, as reference ranges for various age groups, were found to be: 10.63–55.64 pmol/L (20–25 years), 3.74–61.88 pmol/L (26–30 years), 5.49–47.56 pmol/L (31–35 years), 2.15–48.91 pmol/L (36–40 years), 0.92–41.26 pmol/L (41–45 years), and 0.14–5.10 pmol/L (46–50 years).Conclusions: This study (the first in Oman) determined the age-specific reference ranges of serum AMH in healthy Omani women in the age range of 20–50 years

Indigenous marine diatoms as novel sources of bioactive peptides with antihypertensive and antioxidant properties

Several bioactive compounds from microalgae have demonstrated diverse biological activities with positive effects on human health. However, the potential of bioactive peptides as functional foods is still undervalued. Therefore, the exploration of microalgae strains as sources of bioactive peptides could reveal strong and unique bioactivities, especially when these marine sources have never been explored before. For this aim, protein extracts from six indigenous marine diatoms were subjected to enzymatic hydrolysis using four proteases (flavourzyme, pepsin, papain and trypsin). The hydrolysates were then tested for angiotensin converting enzyme (ACE)-inhibitory, antioxidant and antihypertensive properties. Results showed that papain hydrolysates from all microalgae strains exhibited strong ACE-inhibitory activities and antioxidant properties. In particular, protein hydrolysates from Bellerochea malleus were found to reduce blood pressure properties of 17 mmHg after 5 days of oral administration to SHR animals. These results revealed the potential of bioactive peptides from indigenous marine diatoms for use as functional foods or nutraceuticals

The ubiquitin ligase Cullin5^SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2(−/−) mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway

Isolation and identification of indigenous marine diatoms (Bacillariophyta) for biomass production in open raceway ponds

For the successful, large‐scale cultivation of microalgae, which for our work refers to diatoms, it is important to select the appropriate species for the right environment. Intensive research on the growth of different strains of microalgae from several regions worldwide is an ongoing effort. The aim of our research in this context was to select the best isolates of diatoms from different coastal sites in Oman, a region whose flora is poorly known, and identify these species using molecular (nuclear‐encoded small subunit ribosomal RNA and chloroplast‐encoded rbcL and psbC) and morphologic data. We aimed also to investigate and measure the growth rate, biomass production and fatty acid composition under ambient environmental conditions. Our results showed that in contrast to most of the research reported in the literature, the production of biomass from six isolates has been successfully carried out with reduced amounts of nutrients and without CO2 addition. Growth rates for species cultivated outdoors were high and ranged from 0.37 to 0.92 day−1. These findings are economically very promising as fast growth rates, associated with reduced operational costs, could remarkably improve the production efficiency and thereby justify their use as biomass feedstocks. Molecular data revealed that one isolate was Bellerochea malleus, four isolates represented an unidentified species of Bellerochea and the remaining strain studied represented an unidentified species of the genus Nitzschia. These six strains were rich in polyunsaturated fatty acids, with an average of 47% of the total fatty acids, confirming their potential use as aquaculture feed supplements

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

Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

Tuberous sclerosis complex (TSC) is a tumor predisposition syndrome with significant renal cystic and solid tumor disease. While the most common renal tumor in TSC, the angiomyolipoma, exhibits a loss of heterozygosity associated with disease, we have discovered that the renal cystic epithelium is composed of type A intercalated cells that have an intact Tsc gene that have been induced to exhibit Tsc‐mutant disease phenotype. This mechanism appears to be different than that for ADPKD. The murine models described here closely resemble the human disease and both appear to be mTORC1 inhibitor responsive. The induction signaling driving cystogenesis may be mediated by extracellular vesicle trafficking.TSC renal cystic disease develops in about half of the patients. The disease appears to caused by an induction mechanism such that a small population of mutant cells can cause significant renal cystic disease comprised of mostly genetically normal cells.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147796/1/phy213983.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147796/2/phy213983_am.pd

Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system