Joaquín Fernández-Irigoyen

Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics

Primary liver cancer is the fifth most frequent neoplasm and the third most common cause of cancer‐related death, with more than 500 000 new cases diagnosed yearly. The outcome for hepatocellular carcinoma (HCC) patients still remains dismal, partly because of our limited knowledge of its molecular pathogenesis and the difficulty in detecting the disease at its early stages. Therefore, studies aimed at the definition of the mechanisms associated with HCC progression and the identification of new biomarkers leading to early diagnosis and more effective therapeutic interventions are urgently needed. Proteomics is a rapidly expanding discipline that is expected to change the way in which diseases will be diagnosed, treated, and monitored in the near future. In the last few years, HCC has been extensively investigated using different proteomic approaches on HCC cell lines, animal models, and human tumor tissues. In this review, state‐of‐the‐art technology on proteomics is overviewed, and recent advances in liver cancer proteomics and their clinical projections are discussed.

Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer’s disease: identification of common and distinct olfactory targets across Alzheimer-related co-pathologies

Olfactory dysfunction is present in up to 90% of Alzheimers disease (AD) patients. Although deposition of hyperphosphorylated tau and β-amyloid substrates are present in olfactory areas, the molecular mechanisms associated with decreased smell function are not completely understood. We have applied mass spectrometry-based quantitative proteomics to probe additional molecular disturbances in postmortem olfactory bulbs (OB) dissected from AD cases respect to neurologically intact controls (n=20, mean age 82.1 years). Relative proteome abundance measurements have revealed protein interaction networks progressively disturbed across AD stages suggesting an early imbalance in splicing factors, subsequent interrupted cycling of neurotransmitters, alteration in toxic and protective mechanisms of β-amyloid, and finally, a mitochondrial dysfunction together with disturbance in neuron-neuron adhesion. We also present novel molecular findings in the OB in an autopsy cohort composed by Lewy body disease (LBD), frontotemporal lobar degeneration (FTLD), mixed dementia, and progressive supranuclear palsy (PSP) cases (n = 41, mean age 79.7 years). Olfactory mediators deregulated during the progression of AD such as Visinin-like protein 1, RUFY3 protein, and Copine 6 were also differentially modulated in the OB in LBD, FTLD, and mixed dementia. Only Dipeptidyl aminopeptidase-like protein 6 showed a specific down-regulation in AD. However, no differences were observed in the olfactory expression of this protein panel in PSP subjects. This study demonstrates an olfactory progressive proteome modulation in AD, unveiling cross-disease similarities and differences especially for specific proteins involved in dendritic and axonic distributions that occur in the OB during the neurodegenerative process.

Proteomic atlas of the human olfactory bulb

The olfactory bulb (OB) is the first site for the processing of olfactory information in the brain and its deregulation is associated with neurodegenerative disorders. Although different efforts have been made to characterize the human brain proteome in depth, the protein composition of the human OB remains largely unexplored. We have performed a comprehensive analysis of the human OB proteome employing protein and peptide fractionation methods followed by LC-MS/MS, identifying 1529 protein species, corresponding to 1466 unique proteins, which represents a 7-fold increase in proteome coverage with respect to previous OB proteome descriptions from translational models. Bioinformatic analyses revealed that protein components of the OB participated in a plethora of biological process highlighting hydrolase and phosphatase activities and nucleotide and RNA binding activities. Interestingly, 631 OB proteins identified were not previously described in protein datasets derived from large-scale Human Brain Proteome Project (HBPP) studies. In particular, a subset of these differential proteins was mainly involved in axon guidance, opioid signaling, neurotransmitter receptor binding, and synaptic plasticity. Taken together, these results increase our knowledge about the molecular composition of the human OB and may be useful to understand the molecular basis of the olfactory system and the etiology of its disorders.

Enzymatic activity of methionine adenosyltransferase variants identified in patients with persistent hypermethioninemia

Methionine adenosyltransferases (MATs) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal+) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal+ infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca2+-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal+ infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK½, ERK½) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal+ in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

Mass spectrometric characterization of mitochondrial complex I NDUFA10 variants

In the present study, we have used a combination of 2‐DE and MS to isolate and characterize two variants of the mitochondrial complex I subunit NDUFA10 from Wistar rat brain. Extensive MS/MS analysis revealed that a D/N substitution at position 120 resulting from a 353A/G transition in the coding gene is the biochemical difference between the two most abundant NDUFA10 isoforms. Moreover, 33 modifications of distinct chemical nature targeting 59 specific residues were found to be common to the acidic and basic forms. Positions C67, H149 and H322 of NDUFA10 were specially targeted by different modifications suggesting the high reactivity of these residues and their potential implication in the regulation of the protein function. Together with nonenzymatic modifications that can form in the sample isolation and workup steps, such as oxidation of methionine, tryptophan, cysteine and histidine, we describe amino acid variants of unknown chemical structure that must be further characterized, as well as accumulation of R, K and H methylations and probably K acetylations at the C‐terminal region that might play a role in the control of NDUFA10 activity according to similar mechanisms to those described for histones.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea

Highlight A quantitative proteomic approach demonstrates how ammonium nutrition induces glucosinolate biosynthetic and catabolic pathways in Arabidopsis and broccoli.

An early dysregulation of FAK and MEK/ERK signaling pathways precedes the β-amyloid deposition in the olfactory bulb of APP/PS1 mouse model of Alzheimer's disease

UNLABELLED Olfactory dysfunction is an early event of Alzheimers disease (AD). However, the mechanisms associated to AD neurodegeneration in olfactory areas are unknown. Here we used double-transgenic amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) mice and label-free quantitative proteomics to analyze early pathological effects on the olfactory bulb (OB) during AD progression. Prior to β-amyloid plaque formation, 9 modulated proteins were detected on 3-month-old APP/PS1 mice while 16 differential expressed proteins were detected at 6months, when β-amyloid plaques appear, indicating a moderate imbalance in cytoskeletal rearrangement, and synaptic plasticity in APP/PS1 OBs. Moreover, β-amyloid induced an inactivation of focal adhesion kinase (FAK) together with a transient activation of MEK1/2, leading to inactivation of ERK1/2 in 6-months APP/PS1 OBs. In contrast, the analysis of human OBs revealed a late activation of FAK in advanced AD stages, whereas ERK1/2 activation was enhanced across AD staging respect to controls. This survival potential was accompanied by the inhibition of the proapototic factor BAD in the OB across AD phenotypes. Our data contribute to a better understanding of the early molecular mechanisms that are modulated in AD neurodegeneration, highlighting significant differences in the regulation of survival pathways between APP/PS1 mice and sporadic human AD. SIGNIFICANCE Loss of smell is involved in early stages of Alzheimers disease (AD), usually preceding classic disease symptoms. However, the mechanisms governing this dysfunction are still poorly understood, losing its potential as a useful tool for clinical diagnosis. Our study characterizes potential AD-associated molecular changes in APP/PS1 mice olfactory bulb (OB) using MS-quantitative proteomics, revealing early cytoskeletal disruption and synaptic plasticity impairment. Moreover, an opposite pattern was found when comparing the activation status of specific survival pathways between APP/PS1 OBs and OBs derived from sAD subjects with different neuropathological grading. Our data reflect, in part, the progressive effect of APP overproduction and Aβ accumulation on the OB proteome during AD progression.

A core of kinase-regulated interactomes defines the neoplastic MDSC lineage

Myeloid-derived suppressor cells (MDSCs) differentiate from bone marrow precursors, expand in cancer-bearing hosts and accelerate tumor progression. MDSCs have become attractive therapeutic targets, as their elimination strongly enhances anti-neoplastic treatments. Here, immature myeloid dendritic cells (DCs), MDSCs modeling tumor-infiltrating subsets or modeling non-cancerous (NC)-MDSCs were compared by in-depth quantitative proteomics. We found that neoplastic MDSCs differentially expressed a core of kinases which controlled lineage-specific (PI3K-AKT and SRC kinases) and cancer-induced (ERK and PKC kinases) protein interaction networks (interactomes). These kinases contributed to some extent to myeloid differentiation. However, only AKT and ERK specifically drove MDSC differentiation from myeloid precursors. Interfering with AKT and ERK with selective small molecule inhibitors or shRNAs selectively hampered MDSC differentiation and viability. Thus, we provide compelling evidence that MDSCs constitute a distinct myeloid lineage distinguished by a “kinase signature” and well-defined interactomes. Our results define new opportunities for the development of anti-cancer treatments targeting these tumor-promoting immune cells.

MiRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR‐506 is overexpressed in PBC cholangiocytes and directly targets both Cl–/ HCO3− anion exchanger 2 and type III inositol 1,4,5‐trisphosphate receptor, leading to cholestasis. Here, the regulation of miR‐506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin‐8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR‐506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR‐506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR‐506 (1) induced dedifferentiation with down‐regulation of biliary and epithelial markers together with up‐regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase‐3‐dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR‐506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion: Different proinflammatory cytokines enhance the expression of miR‐506 in biliary epithelial cells; miR‐506 induces PBC‐like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420‐1440)