Pathogen-driven degradation of endogenous and therapeutic antibodies during streptococcal infections.

Group A streptococcus (GAS) is a major bacterial pathogen responsible for both local and systemic infections in humans. The molecular mechanisms that contribute to disease heterogeneity remain poorly understood. Here we show that the transition from a local to a systemic GAS infection is paralleled by pathogen-driven alterations in IgG homeostasis. Using animal models and a combination of sensitive proteomics and glycoproteomics readouts, we documented the progressive accumulation of IgG cleavage products in plasma, due to extensive enzymatic degradation triggered by GAS infection in vivo. The level of IgG degradation was modulated by the route of pathogen inoculation, and mechanistically linked to the combined activities of the bacterial protease IdeS and the endoglycosidase EndoS, upregulated during infection. Importantly, we show that these virulence factors can alter the structure and function of exogenous therapeutic IgG in vivo. These results shed light on the role of bacterial virulence factors in shaping GAS pathogenesis, and potentially blunting the efficacy of antimicrobial therapies.

Proteomic analysis across patient iPSC-based models and human post-mortem hippocampal tissue reveals early cellular dysfunction and progression of Alzheimer's disease pathogenesis.

The hippocampus is a primary region affected in Alzheimer's disease (AD). Because AD postmortem brain tissue is not available prior to symptomatic stage, we lack understanding of early cellular pathogenic mechanisms. To address this issue, we examined the cellular origin and progression of AD pathogenesis by comparing patient-based model systems including iPSC-derived brain cells transplanted into the mouse brain hippocampus. Proteomic analysis of the graft enabled the identification of pathways and network dysfunction in AD patient brain cells, associated with increased levels of Aß-42 and ß-sheet structures. Interestingly, the host cells surrounding the AD graft also presented alterations in cellular biological pathways. Furthermore, proteomic analysis across human iPSC-based models and human post-mortem hippocampal tissue projected coherent longitudinal cellular changes indicative of early to end stage AD cellular pathogenesis. Our data showcase patient-based models to study the cell autonomous origin and progression of AD pathogenesis.

Proteomic profiles of Zika virus-infected placentas bearing fetuses with microcephaly.

PURPOSE: Zika virus (ZIKV) transmission to the fetus during pregnancy could enable a collection of severe fetal malformations like microcephaly (MC), termed Congenital Zika Syndrome (CZS). The mechanisms involved in ZIKV transplacental transmission are not fully understood. EXPERIMENTAL DESIGN: Here we aim to identify in placental tissues the deregulated proteins associated with ZIKV-induced MC using label-free proteomics. RESULTS: We found proteins associated with DNA damage and gene expression inhibition up-regulated in infected placentas with no MC fetuses (Z+) compared to the control group (Ctr). Actin filament organization and the immune response were also found deregulated in the Z+ group. In ZIKV-positive placentas bearing fetuses with MC (MC+) was detected an increase in T cell activation, indicating an elevated immune response. A comparison between MC+ and Z+ groups showed a higher abundance of proteins related to endocytosis and autophagy in MC+, suggesting a higher transcytosis of vesicles with ZIKV particles across the maternal-fetal interface. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that higher expression of integrins in MC+ might be associated with high internalization of the virus since these proteins are known as virus receptors. Similarly, an increased immune response in the placenta and higher infiltration of the virus to the fetus could contribute to the neurological malformation of the CZS.

Deep Proteomic Analysis on Biobanked Paraffine-Archived Melanoma with Prognostic/Predictive Biomarker Read-Out.

The discovery of novel protein biomarkers in melanoma is crucial. Our introduction of formalin-fixed paraffin-embedded (FFPE) tumor protocol provides new opportunities to understand the progression of melanoma and open the possibility to screen thousands of FFPE samples deposited in tumor biobanks and available at hospital pathology departments. In our retrospective biobank pilot study, 90 FFPE samples from 77 patients were processed. Protein quantitation was performed by high-resolution mass spectrometry and validated by histopathologic analysis. The global protein expression formed six sample clusters. Proteins such as TRAF6 and ARMC10 were upregulated in clusters with enrichment for shorter survival, and proteins such as AIFI1 were upregulated in clusters with enrichment for longer survival. The cohort's heterogeneity was addressed by comparing primary and metastasis samples, as well comparing clinical stages. Within immunotherapy and targeted therapy subgroups, the upregulation of the VEGFA-VEGFR2 pathway, RNA splicing, increased activity of immune cells, extracellular matrix, and metabolic pathways were positively associated with patient outcome. To summarize, we were able to (i) link global protein expression profiles to survival, and they proved to be an independent prognostic indicator, as well as (ii) identify proteins that are potential predictors of a patient's response to immunotherapy and targeted therapy, suggesting new opportunities for precision medicine developments.

Topological Dissection of Proteomic Changes Linked to the Limbic Stage of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia worldwide. In AD, neurodegeneration spreads throughout different areas of the central nervous system (CNS) in a gradual and predictable pattern, causing progressive memory decline and cognitive impairment. Deposition of neurofibrillary tangles (NFTs) in specific CNS regions correlates with the severity of AD and constitutes the basis for disease classification into different Braak stages (I-VI). Early clinical symptoms are typically associated with stages III-IV (i.e., limbic stages) when the involvement of the hippocampus begins. Histopathological changes in AD have been linked to brain proteome alterations, including aberrant posttranslational modifications (PTMs) such as the hyperphosphorylation of Tau. Most proteomic studies to date have focused on AD progression across different stages of the disease, by targeting one specific brain area at a time. However, in AD vulnerable regions, stage-specific proteomic alterations, including changes in PTM status occur in parallel and remain poorly characterized. Here, we conducted proteomic, phosphoproteomic, and acetylomic analyses of human postmortem tissue samples from AD (Braak stage III-IV, n=11) and control brains (n=12), covering all anatomical areas affected during the limbic stage of the disease (total hippocampus, CA1, entorhinal and perirhinal cortices). Overall, ~6000 proteins, ~9000 unique phosphopeptides and 221 acetylated peptides were accurately quantified across all tissues. Our results reveal significant proteome changes in AD brains compared to controls. Among others, we have observed the dysregulation of pathways related to the adaptive and innate immune responses, including several altered antimicrobial peptides (AMPs). Notably, some of these changes were restricted to specific anatomical areas, while others altered according to disease progression across the regions studied. Our data highlights the molecular heterogeneity of AD and the relevance of neuroinflammation as a major player in AD pathology. Data are available via ProteomeXchange with identifier PXD027173.

The Human Melanoma Proteome Atlas-Complementing the melanoma transcriptome.

The MM500 meta-study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass-spectrometry-based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well-annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein-coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease.

The human melanoma proteome atlas-Defining the molecular pathology.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in-depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.

Molecular alterations in the extracellular matrix in the brains of newborns with congenital Zika syndrome.

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.

The Role of Microglia in Glioblastoma.

Glioblastoma (GB), the most aggressive malignant glioma, is made up of a large percentage of glioma-associated microglia/macrophages (GAM), suggesting that immune cells play an important role in the pathophysiology of GB. Under physiological conditions, microglia, the phagocytes of the central nervous system (CNS), are involved in various processes such as neurogenesis or axonal growth, and the progression of different conditions such as Alzheimer's disease. Through immunohistochemical studies, markers that enhance GB invasiveness have been shown to be expressed in the peritumoral area of ​​the brain, such as Transforming Growth Factor α (TGF-α), Stromal Sell-Derived Factor 1 (SDF1/CXCL12), Sphingosine-1-Phosphate (S1P) and Neurotrophic Factor Derived from the Glial cell line (GDNF), contributing to the increase in tumor mass. Similarly, it has also been described 17 biomarkers that are present in hypoxic periarteriolar HSC niches in bone marrow and in hypoxic periarteriolar GSC niches in glioblastoma. Interestingly, microglia plays an important role in the microenvironment that supports GB progression, being one of the most important focal points in the study of therapeutic targets for the development of new drugs. In this review, we describe the altered signaling pathways in microglia in the context of GB. We also show how microglia interact with glioblastoma cells and the epigenetic mechanisms involved. Regarding the interactions between microglia and neurogenic niches, some authors indicate that glioblastoma stem cells (GSC) are similar to neural stem cells (NSC), common stem cells in the subventricular zone (SVZ), suggesting that this could be the origin of GB. Understanding the similarities between SVZ and the tumor microenvironment could be important to clarify some mechanisms involved in GB malignancy and to support the discovering of new therapeutic targets for the development of more effective glioblastoma treatments.

Proteome Dynamics of the Developing Açaí Berry Pericarp (Euterpe oleracea Mart.).

Quantitative proteome analysis of four developmental stages of pericarp tissues of the açaí berry (Euterpe oleracea Mart.) was performed by the isobaric labeling of peptides with iTRAQ 4-plex, hydrophilic interaction liquid chromatography pre-fractionation of labeled peptides, and high-performance mass spectrometry analysis. This analysis resulted in the identification of 4286 proteins, of which 476 presented differential abundance between the stages. The differential abundance of these proteins was seen to be coordinated with the metabolic demands during cell division, lignification, and cell expansion at developmental stages 1 and 2 as well as phenolic acid accumulation and metabolic changes in the fruit maturation at developmental stages 3 and 4. The distinct accumulation of anthocyanins observed in the pericarp at developmental stage 4 was correlated with the increase in abundance of some key biosynthetic enzymes, such as leucoanthocyanidin dioxygenase, anthocyanidin O-3-glycosyltransferase, and UDP-glycosyltransferase. Here, evidence is also provided for the presence in the açaí berry of secondary metabolites not previously described in açaí, such as pterostilbene, matairesinol, and furaneol. Together, these results will pave the way for studies aimed at the genetic improvement of the nutritional properties of this important fruit crop.

Proteomic analysis of whole saliva in chronic periodontitis.

Periodontitis is a chronic inflammatory disease resulting from a dysbiosis of the dental biofilm and a dysregulated host response in susceptible individuals. It is characterized by periodontal attachment destruction, bone resorption and eventual tooth loss. Salivary biomarkers have been sought to predict and prevent periodontitis. This comparative study analyzed the salivary proteome of individuals with chronic periodontitis (CP) and periodontal health (PH) and correlated specific proteins with clinical parameters of disease by using mass spectrometry. Stimulated whole saliva was obtained 10 PH and 30 CP patients and pooled into 5 healthy control samples and 15 CP samples. After precipitation with TCA, samples were digested enzymatically with trypsin and analyzed by a LTQ Orbitrap Velos equipped with a nanoelectrospray ion source. A wide range of salivary proteins of various functions was significantly reduced in CP individuals, whereas salivary acidic proline-rich phosphoprotein, submaxillary gland androgen-regulated protein, histatin-1, fatty acid binding protein, thioredoxin and cystatin-SA were predominant in diseased patients and correlated significantly with signs of periodontal attachment loss and inflammation. In conclusion, few specific salivary proteins were associated with CP. These findings may contribute to the identification of disease indicators or signatures for the improvement of periodontal diagnosis. SIGNIFICANCE: Periodontitis is a chronic inflammatory disease that results in periodontal attachment destruction, bone resorption and eventual tooth loss. Salivary biomarkers have been sought to predict periodontitis. The analysis of the salivary proteome of individuals with chronic periodontitis indicated that several proteins of various functions were significantly reduced in these individuals, except for salivary acidic proline-rich phosphoprotein, submaxillary gland androgen-regulated protein, histatin, fatty acid binding protein, thioredoxin and cystatin. Differences in salivary proteome profiles between periodontal health and periodontitis may contribute to the identification of disease indicators and to the improvement of periodontal diagnosis and treatment.

Molecular alterations in the extracellular matrix in the brains of newborns with congenital Zika syndrome

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.

Molecular alterations in the extracellular matrix in the brains of newborns with congenital Zika syndrome

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.

Quantitative Subcellular Proteomics of the Orbitofrontal Cortex of Schizophrenia Patients.

Schizophrenia is a chronic disease characterized by the impairment of mental functions with a marked social dysfunction. A quantitative proteomic approach using iTRAQ labeling and SRM, applied to the characterization of mitochondria (MIT), crude nuclear fraction (NUC), and cytoplasm (CYT), can allow the observation of dynamic changes in cell compartments providing valuable insights concerning schizophrenia physiopathology. Mass spectrometry analyses of the orbitofrontal cortex from 12 schizophrenia patients and 8 healthy controls identified 655 protein groups in the MIT fraction, 1500 in NUC, and 1591 in CYT. We found 166 groups of proteins dysregulated among all enriched cellular fractions. Through the quantitative proteomic analysis, we detect as the main biological pathways those related to calcium and glutamate imbalance, cell signaling disruption of CREB activation, axon guidance, and proteins involved in the activation of NF-kB signaling along with the increase of complement protein C3. Based on our data analysis, we suggest the activation of NF-kB as a possible pathway that links the deregulation of glutamate, calcium, apoptosis, and the activation of the immune system in schizophrenia patients. All MS data are available in the ProteomeXchange Repository under the identifier PXD015356 and PXD014350.

Synaptosomal Proteome of the Orbitofrontal Cortex from Schizophrenia Patients Using Quantitative Label-Free and iTRAQ-Based Shotgun Proteomics.

Schizophrenia is a chronic and incurable neuropsychiatric disorder that affects about one percent of the world population. The proteomic characterization of the synaptosome fraction of the orbitofrontal cortex is useful for providing valuable information about the molecular mechanisms of synaptic functions in these patients. Quantitative analyses of synaptic proteins were made with eight paranoid schizophrenia patients and a pool of eight healthy controls free of mental diseases. Label-free and iTRAQ labeling identified a total of 2018 protein groups. Statistical analyses revealed 12 and 55 significantly dysregulated proteins by iTRAQ and label-free, respectively. Quantitative proteome analyses showed an imbalance in the calcium signaling pathway and proteins such as reticulon-1 and cytochrome c, related to endoplasmic reticulum stress and programmed cell death. Also, it was found that there is a significant increase in limbic-system-associated membrane protein and α-calcium/calmodulin-dependent protein kinase II, associated with the regulation of human behavior. Our data contribute to a better understanding about apoptosis as a possible pathophysiological mechanism of this disease as well as neural systems supporting social behavior in schizophrenia. This study also is a joint effort of the Chr 15 C-HPP team and the Human Brain Proteome Project of B/D-HPP. All MS proteomics data are deposited in the ProteomeXchange Repository under PXD006798.

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.