Publications by authors named "James D Sutherland"

54 Publications

SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes.

Front Cell Dev Biol 2021 21;9:715868. Epub 2021 Sep 21.

Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance, Derio, Spain.

Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development. Both proteins localize to nuclear bodies and are modified by the small ubiquitin-like modifier (SUMO). Our results show that CBX4 and SALL1 interact in the nucleoplasm and that increased SALL1 expression reduces ubiquitination of CBX4, enhancing its stability. This is accompanied by an increase in the number and size of CBX4-containing Polycomb bodies, and by a greater repression of CBX4 target genes. Thus, our findings uncover a new way of SALL1-mediated regulation of Polycomb bodies through modulation of CBX4 stability, with consequences in the regulation of its target genes, which could have an impact in cell differentiation and development.
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http://dx.doi.org/10.3389/fcell.2021.715868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490708PMC
September 2021

High prevalence of sternal foramina in indigenous Bolivians compared to Midwest Americans and indigenous North Americans (sternal foramina in indigenous Bolivians).

Anat Sci Int 2021 Sep 30;96(4):517-523. Epub 2021 May 30.

St. Luke's Mid-America Heart Institute of Kansas City, 4330 Wornall Rd., Suite 2000, Kansas City, MO, 64111, USA.

The sternal foramen, usually an asymptomatic osteological defect, can lead to catastrophic consequences if not recognized prior to certain medical procedures. This study reports the prevalence of a sternal foramen in two South Amerindian populations compared with other published populations. We evaluated the presence of sternal foramina using thoracic computed tomography scans of 1334 (48% female) participants from two indigenous populations of Bolivia (n = 900 Tsimane, 434 Moseten). The prevalence of sternal foramina was compared to two U.S. populations of similar sex/age distribution (n = 572 Midwest Americans, 131 self-identified Native North Americans) via similar CT scans. A sternal foramen was significantly more common in the two Bolivian populations (prevalence ranging from 12.8 to 13.4%), compared to 4.4-5.1% in the two U.S. groups, consistent with prior estimates in studies from industrialized populations. Males had higher frequency of a sternal foramen compared to females in each of the four groups (OR = 1.904, 95% CI: 1.418-2.568, p < 0.001). Age was not associated with sternal foramen presence. These data show both a higher rate of sternal foramina in the South Amerindian populations versus comparator populations in North America and the highest rate of any studied living population. Although it is not possible to determine from our data the relative contribution of genetics versus early life or environmental causes to the higher rates of sternal foramen, we note that small prior studies have likewise demonstrated a higher prevalence in lower income countries. Further determination of the contributing factors warrants greater investigation and research.
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http://dx.doi.org/10.1007/s12565-021-00618-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445587PMC
September 2021

The indigenous South American Tsimane exhibit relatively modest decrease in brain volume with age despite high systemic inflammation.

J Gerontol A Biol Sci Med Sci 2021 May 26. Epub 2021 May 26.

Economic Science Institute, Argyros School of Business and Economics, Chapman University, 1 University Drive, Orange CA 92866 USA.

Brain atrophy is correlated with risk of cognitive impairment, functional decline, and dementia. Despite a high infectious disease burden, Tsimane forager-horticulturists of Bolivia have the lowest prevalence of coronary atherosclerosis of any studied population and present few cardiovascular disease (CVD) risk factors despite a high burden of infections and therefore inflammation. This study (A) examines the statistical association between brain volume and age for Tsimane, and (B) compares this association to that of three industrialized populations in the U.S. and Europe. This cohort-based panel study enrolled 746 participants aged 40 to 94 (396 males), from whom computed tomography (CT) head scans were acquired. Brain volume (BV) and intracranial volume (ICV) were calculated from automatic head CT segmentations. The linear regression coefficient estimate β⌢T of the Tsimane (T), describing the relationship between age (predictor) and BV (response, as a percentage of ICV), was calculated for the pooled sample (including both sexes) and for each sex. β⌢T was compared to the corresponding regression coefficient estimate β⌢R of samples from the industrialized reference (R) countries. For all comparisons, the null hypothesis βT = βR was rejected both for the combined samples of males and females, as well as separately for each sex. Our results indicate that the Tsimane exhibit a significantly slower decrease in brain volume with age than populations in the U.S. and Europe. Such reduced rates of brain volume decrease, together with a subsistence lifestyle and low cardiovascular disease risk, may protect brain health despite considerable chronic inflammation related to infectious burden.
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http://dx.doi.org/10.1093/gerona/glab138DOI Listing
May 2021

LUZP1 Controls Cell Division, Migration and Invasion Through Regulation of the Actin Cytoskeleton.

Front Cell Dev Biol 2021 1;9:624089. Epub 2021 Apr 1.

Center for Cooperative Research in Biosciences (CIC BioGUNE), Basque Research and Technology Alliance, Bizkaia Technology Park, Derio, Spain.

LUZP1 is a centrosomal and actin cytoskeleton-localizing protein that regulates both ciliogenesis and actin filament bundling. As the cytoskeleton and cilia are implicated in metastasis and tumor suppression, we examined roles for LUZP1 in the context of cancer. Here we show that exhibits frequent genomic aberrations in cancer, with a predominance of gene deletions. Furthermore, we demonstrate that CRISPR/Cas9-mediated loss of in mouse fibroblasts promotes cell migration and invasion features, reduces cell viability, and increases cell apoptosis, centriole numbers, and nuclear size while altering the actin cytoskeleton. Loss of also induced changes to ACTR3 (Actin Related Protein 3, also known as ARP3) and phospho-cofilin ratios, suggesting regulatory roles in actin polymerization, beyond its role in filament bundling. Our results point to an unprecedented role for LUZP1 in the regulation of cancer features through the control of actin cytoskeleton.
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http://dx.doi.org/10.3389/fcell.2021.624089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049182PMC
April 2021

Virtual meeting, real and sound science: report of the 17 Meeting of the Spanish Society for Developmental Biology (SEBD-2020).

Int J Dev Biol 2021 ;65(7-8-9):457-464

Department of Genetics, Microbiology and Statistics, and Institute of Biomedicine (IBUB), School of Biology, University of Barcelona, Barcelona, Spain.

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.
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http://dx.doi.org/10.1387/ijdb.210005rbDOI Listing
October 2021

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl-Induced Liver Fibrosis.

Int J Mol Sci 2020 Nov 27;21(23). Epub 2020 Nov 27.

Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), 48160 Derio, Bizkaia, Spain.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process "protein polyubiquitination" is enriched after CCl-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (Ub mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.
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http://dx.doi.org/10.3390/ijms21239043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729774PMC
November 2020

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is a contributing factor in Townes-Brocks Syndrome.

Elife 2020 06 18;9. Epub 2020 Jun 18.

Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain.

Primary cilia are sensory organelles crucial for cell signaling during development and organ homeostasis. Cilia arise from centrosomes and their formation and function is governed by numerous factors. Through our studies on Townes-Brocks Syndrome (TBS), a rare disease linked to abnormal cilia formation in human fibroblasts, we uncovered the leucine-zipper protein LUZP1 as an interactor of truncated SALL1, a dominantly-acting protein causing the disease. Using TurboID proximity labeling and pulldowns, we show that LUZP1 associates with factors linked to centrosome and actin filaments. Here, we show that LUZP1 is a cilia regulator. It localizes around the centrioles and to actin cytoskeleton. Loss of LUZP1 reduces F-actin levels, facilitates ciliogenesis and alters Sonic Hedgehog signaling, pointing to a key role in cytoskeleton-cilia interdependency. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Together with other factors, alterations in LUZP1 may be contributing to TBS etiology.
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http://dx.doi.org/10.7554/eLife.55957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363444PMC
June 2020

HuR/ELAVL1 drives malignant peripheral nerve sheath tumor growth and metastasis.

J Clin Invest 2020 07;130(7):3848-3864

Department of Pathology, University of Chicago, Chicago, Illinois, USA.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/β-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment.
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http://dx.doi.org/10.1172/JCI130379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324187PMC
July 2020

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer.

J Exp Med 2020 06;217(6)

The Institute of Cancer Research, London, UK.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.
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http://dx.doi.org/10.1084/jem.20191787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971141PMC
June 2020

Regulation of the Ebola Virus VP24 Protein by SUMO.

J Virol 2019 12 12;94(1). Epub 2019 Dec 12.

Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione -transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein. The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.
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http://dx.doi.org/10.1128/JVI.01687-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912094PMC
December 2019

Targeting PML in triple negative breast cancer elicits growth suppression and senescence.

Cell Death Differ 2020 04 1;27(4):1186-1199. Epub 2019 Oct 1.

CIC bioGUNE, Derio, Spain.

Oncogene addiction postulates that the survival and growth of certain tumor cells is dependent upon the activity of one oncogene, despite their multiple genetic and epigenetic abnormalities. This phenomenon provides a foundation for molecular targeted therapy and a rationale for oncogene-based stratification. We have previously reported that the Promyelocytic Leukemia protein (PML) is upregulated in triple negative breast cancer (TNBC) and it regulates cancer-initiating cell function, thus suggesting that this protein can be therapeutically targeted in combination with PML-based stratification. However, the effects of PML perturbation on the bulk of tumor cells remained poorly understood. Here we demonstrate that TNBC cells are addicted to the expression of this nuclear protein. PML inhibition led to a remarkable growth arrest combined with features of senescence in vitro and in vivo. Mechanistically, the growth arrest and senescence were associated to a decrease in MYC and PIM1 kinase levels, with the subsequent accumulation of CDKN1B (p27), a trigger of senescence. In line with this notion, we found that PML is associated to the promoter regions of MYC and PIM1, consistent with their direct correlation in breast cancer specimens. Altogether, our results provide a feasible explanation for the functional similarities of MYC, PIM1, and PML in TNBC and encourage further study of PML targeting strategies for the treatment of this breast cancer subtype.
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http://dx.doi.org/10.1038/s41418-019-0407-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104349PMC
April 2020

Proteostasis: the network behind the networks.

Semin Cell Dev Biol 2019 May 9. Epub 2019 May 9.

CIC bioGUNE, Bilbao, Spain(1).

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http://dx.doi.org/10.1016/j.semcdb.2019.05.005DOI Listing
May 2019

Proteostasis: The network behind the networks.

Semin Cell Dev Biol 2019 09 21;93:97-99. Epub 2019 Apr 21.

CIC bioGUNE, Bilbao, Spain(1). Electronic address:

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http://dx.doi.org/10.1016/j.semcdb.2019.04.011DOI Listing
September 2019

Putting the Stress on UFM1 (Ubiquitin-Fold Modifier 1).

Circ Heart Fail 2018 10;11(10):e005455

CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.

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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.118.005455DOI Listing
October 2018

Truncated SALL1 Impedes Primary Cilia Function in Townes-Brocks Syndrome.

Am J Hum Genet 2018 02;102(2):249-265

CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 Derio, Bizkaia, Spain. Electronic address:

Townes-Brocks syndrome (TBS) is characterized by a spectrum of malformations in the digits, ears, and kidneys. These anomalies overlap those seen in a growing number of ciliopathies, which are genetic syndromes linked to defects in the formation or function of the primary cilia. TBS is caused by mutations in the gene encoding the transcriptional repressor SALL1 and is associated with the presence of a truncated protein that localizes to the cytoplasm. Here, we provide evidence that SALL1 mutations might cause TBS by means beyond its transcriptional capacity. By using proximity proteomics, we show that truncated SALL1 interacts with factors related to cilia function, including the negative regulators of ciliogenesis CCP110 and CEP97. This most likely contributes to more frequent cilia formation in TBS-derived fibroblasts, as well as in a CRISPR/Cas9-generated model cell line and in TBS-modeled mouse embryonic fibroblasts, than in wild-type controls. Furthermore, TBS-like cells show changes in cilia length and disassembly rates in combination with aberrant SHH signaling transduction. These findings support the hypothesis that aberrations in primary cilia and SHH signaling are contributing factors in TBS phenotypes, representing a paradigm shift in understanding TBS etiology. These results open possibilities for the treatment of TBS.
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http://dx.doi.org/10.1016/j.ajhg.2017.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985538PMC
February 2018

PPARδ Elicits Ligand-Independent Repression of Trefoil Factor Family to Limit Prostate Cancer Growth.

Cancer Res 2018 01 29;78(2):399-409. Epub 2017 Nov 29.

CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.

The nuclear receptor PPAR-β/δ (PPARD) has essential roles in fatty acid catabolism and energy homeostasis as well as cell differentiation, inflammation, and metabolism. However, its contributions to tumorigenesis are uncertain and have been disputed. Here, we provide evidence of tumor suppressive activity of PPARD in prostate cancer through a noncanonical and ligand-independent pathway. PPARD was downregulated in prostate cancer specimens. In murine prostate epithelium, PPARD gene deletion resulted in increased cellularity. Genetic modulation of PPARD in human prostate cancer cell lines validated the tumor suppressive activity of this gene and Mechanistically, PPARD exerted its activity in a DNA binding-dependent and ligand-independent manner. We identified a novel set of genes repressed by PPARD that failed to respond to ligand-mediated activation. Among these genes, we observed robust regulation of the secretory trefoil factor family (TFF) members, including a causal and correlative association of TFF1 with prostate cancer biology and in patient specimens. Overall, our results illuminate the oncosuppressive function of PPARD and understanding of the pathogenic molecular pathways elicited by this nuclear receptor. These findings challenge the presumption that the function of the nuclear receptor PPARβ/δ in cancer is dictated by ligand-mediated activation. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0908DOI Listing
January 2018

The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant.

Sci Rep 2017 09 6;7(1):10740. Epub 2017 Sep 6.

CIC bioGUNE, 48160, Derio, Bizkaia, Spain.

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.
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http://dx.doi.org/10.1038/s41598-017-11354-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587732PMC
September 2017

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Nature 2017 07 28;547(7661):109-113. Epub 2017 Jun 28.

CIC bioGUNE, Bizkaia Technology Park, 801 building, 48160, Derio, Spain.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.
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http://dx.doi.org/10.1038/nature22964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505479PMC
July 2017

Is coronary calcium scoring too late? Total body arterial calcium burden in patients without known CAD and normal MPI.

J Nucl Cardiol 2018 12 25;25(6):1990-1998. Epub 2017 May 25.

Ascension Healthcare, Milwaukee, WI, USA.

Background: Patients with normal myocardial perfusion imaging (MPI) have a good prognosis. However, pre-clinical coronary and extracoronary atherosclerosis may exist in the absence of myocardial ischemia.

Methods: 154 Egyptian patients (mean age 53 years) underwent whole-body non-contrast CT following normal MPI.

Results: Atherosclerosis in the form of calcification was observed in ≥1 vascular bed in 115 of 154 (75%) patients. This included the iliofemoral (62%), abdominal aorta (53%), thoracic aorta (47%), coronary (47%), and carotid (25%) vascular beds. Mean total body calcium score was 3172 ± 530 AU. Extracoronary atherosclerosis in patients with a zero coronary artery calcium (CAC) score was common, occurring in the above-listed beds 42%, 36%, 29%, and 7% of the time, respectively. CAC was rarely present without iliofemoral or abdominal aortic calcification.

Conclusion: Quantitative assessment of calcification in different vascular beds demonstrates that extracoronary atherosclerosis is common in patients who have normal MPI. Atherosclerotic calcifications are most common in the iliofemoral arteries and abdominal aorta, which typically predate coronary calcifications. An imaging strategy to detect extracoronary atherosclerosis could lead to greater understanding of the natural history of atherosclerosis in its long pre-clinical phase and possibly to earlier preventive strategies.
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http://dx.doi.org/10.1007/s12350-017-0925-9DOI Listing
December 2018

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

Mol Neurodegener 2017 04 11;12(1):29. Epub 2017 Apr 11.

Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.

Background: Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.

Methods: Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.

Results: We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.

Conclusions: Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.
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http://dx.doi.org/10.1186/s13024-017-0170-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387213PMC
April 2017

Coronary atherosclerosis in indigenous South American Tsimane: a cross-sectional cohort study.

Lancet 2017 04 17;389(10080):1730-1739. Epub 2017 Mar 17.

University of California Irvine, Orange, CA, USA.

Background: Conventional coronary artery disease risk factors might potentially explain at least 90% of the attributable risk of coronary artery disease. To better understand the association between the pre-industrial lifestyle and low prevalence of coronary artery disease risk factors, we examined the Tsimane, a Bolivian population living a subsistence lifestyle of hunting, gathering, fishing, and farming with few cardiovascular risk factors, but high infectious inflammatory burden.

Methods: We did a cross-sectional cohort study including all individuals who self-identified as Tsimane and who were aged 40 years or older. Coronary atherosclerosis was assessed by coronary artery calcium (CAC) scoring done with non-contrast CT in Tsimane adults. We assessed the difference between the Tsimane and 6814 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). CAC scores higher than 100 were considered representative of significant atherosclerotic disease. Tsimane blood lipid and inflammatory biomarkers were obtained at the time of scanning, and in some patients, longitudinally.

Findings: Between July 2, 2014, and Sept 10, 2015, 705 individuals, who had data available for analysis, were included in this study. 596 (85%) of 705 Tsimane had no CAC, 89 (13%) had CAC scores of 1-100, and 20 (3%) had CAC scores higher than 100. For individuals older than age 75 years, 31 (65%) Tsimane presented with a CAC score of 0, and only four (8%) had CAC scores of 100 or more, a five-fold lower prevalence than industrialised populations (p≤0·0001 for all age categories of MESA). Mean LDL and HDL cholesterol concentrations were 2·35 mmol/L (91 mg/dL) and 1·0 mmol/L (39·5 mg/dL), respectively; obesity, hypertension, high blood sugar, and regular cigarette smoking were rare. High-sensitivity C-reactive protein was elevated beyond the clinical cutoff of 3·0 mg/dL in 360 (51%) Tsimane participants.

Interpretation: Despite a high infectious inflammatory burden, the Tsimane, a forager-horticulturalist population of the Bolivian Amazon with few coronary artery disease risk factors, have the lowest reported levels of coronary artery disease of any population recorded to date. These findings suggest that coronary atherosclerosis can be avoided in most people by achieving a lifetime with very low LDL, low blood pressure, low glucose, normal body-mass index, no smoking, and plenty of physical activity. The relative contributions of each are still to be determined.

Funding: National Institute on Aging, National Institutes of Health; St Luke's Hospital of Kansas City; and Paleocardiology Foundation.
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http://dx.doi.org/10.1016/S0140-6736(17)30752-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028773PMC
April 2017

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation.

Sci Rep 2017 01 18;7:40756. Epub 2017 Jan 18.

CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.
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http://dx.doi.org/10.1038/srep40756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241687PMC
January 2017

Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy.

Methods Mol Biol 2016 ;1475:161-9

CIC bioGUNE, Bizkaia Technology Park, Derio, Bizkaia, 48160, Spain.

Posttranslational regulation of proteins by conjugation of ubiquitin- and ubiquitin-like molecules is a common theme in almost every known biological pathway. SUMO (small ubiquitin-related modifier) is dynamically added and deleted from many cellular substrates to control activity, localization, and recruitment of other SUMO-recognizing protein complexes. The dynamic nature of this modification and its low abundance in resting cells make it challenging to study, with susceptibility to deSUMOylases further complicating its analysis. Here we describe bioSUMO, a general method to isolate and analyze SUMOylated proteins from cultured cells, using Drosophila as a highlighted example. The method also has been validated in transgenic flies, as well as human cells. SUMOylated substrates are labeled by in vivo biotinylation, which facilitates their subsequent purification using streptavidin-based affinity chromatography under stringent conditions and with very low background. The bioSUMO approach can be used to validate whether a specific protein is modified, or used to analyze an entire SUMO subproteome. If coupled to quantitative proteomics methods, it may reveal how the SUMO landscape changes with different stimuli, or in diverse cell or tissue types. This technique offers a complementary approach to study SUMO biology and we expect that the strategy can be extended to other ubiquitin-like proteins.
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http://dx.doi.org/10.1007/978-1-4939-6358-4_12DOI Listing
January 2018

Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins.

Methods Mol Biol 2016 ;1475:109-21

INBIOMED, San Sebastian, Spain.

SUMO-interacting motifs (SIMs) recognize SUMOylated proteins with high specificity allowing to connect SUMO-modified proteins. Multiple SIMs fused to distinct tags have been used to increase their affinity and generate more efficient purification tools. Enrichment of SUMOylated proteins using SIMs arranged in tandem (SUMO-traps) facilitates the identification and characterization of protein targets in vitro and in vivo. Here a protocol to produce biotinylated SUMO-traps (bioSUBEs) to capture SUMO chains and typical SUMOylated proteins such as p53 or IkBα is presented. Biotinylated SUMO-traps represent an alternative to reduce the background associated to bigger tags, e.g., during mass spectrometry analysis. Consequently, bioSUBEs are alternative tools to characterize endogenous SUMO targets.
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http://dx.doi.org/10.1007/978-1-4939-6358-4_8DOI Listing
January 2018

Stratification and therapeutic potential of PML in metastatic breast cancer.

Nat Commun 2016 08 24;7:12595. Epub 2016 Aug 24.

CIC bioGUNE, Bizkaia Technology Park, Bulding 801a, 48160 Derio, Spain.

Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification.
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http://dx.doi.org/10.1038/ncomms12595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999521PMC
August 2016

Transcriptomic profiling of urine extracellular vesicles reveals alterations of CDH3 in prostate cancer.

Oncotarget 2016 Feb;7(6):6835-46

CIC bioGUNE, Bizkaia Technology Park, Biscay, Spain.

Extracellular vesicles (EV) are emerging structures with promising properties for intercellular communication. In addition, the characterization of EV in biofluids is an attractive source of non-invasive diagnostic, prognostic and predictive biomarkers. Here we show that urinary EV (uEV) from prostate cancer (PCa) patients exhibit genuine and differential physical and biological properties compared to benign prostate hyperplasia (BPH). Importantly, transcriptomics characterization of uEVs led us to define the decreased abundance of Cadherin 3, type 1 (CDH3) transcript in uEV from PCa patients. Tissue and cell line analysis strongly suggested that the status of CDH3 in uEVs is a distal reflection of changes in the expression of this cadherin in the prostate tumor. CDH3 was negatively regulated at the genomic, transcriptional, and epigenetic level in PCa. Our results reveal that uEVs could represent a non-invasive tool to inform about the molecular alterations in PCa.
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http://dx.doi.org/10.18632/oncotarget.6899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872752PMC
February 2016

Evolution of SUMO Function and Chain Formation in Insects.

Mol Biol Evol 2016 Feb 4;33(2):568-84. Epub 2015 Nov 4.

Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.
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http://dx.doi.org/10.1093/molbev/msv242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866545PMC
February 2016

Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

Sci Rep 2015 Sep 30;5:14692. Epub 2015 Sep 30.

Department of Veterinary and Animal Sciences. University of Massachusetts Amherst. Amherst, MA 01003.

MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.
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http://dx.doi.org/10.1038/srep14692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588509PMC
September 2015
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