Conditional essentiality of the 11-subunit complex I-like enzyme in strict anaerobes: the case of Desulfitobacterium hafniense strain DCB-2.

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

Phosphoproteomics and morphology of stored human red blood cells treated by protein tyrosine phosphatases inhibitor.

ABSTRACT: The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. This study investigated the relationship between pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate [OV]) to stimulate phosphorylation and with 3 selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914) and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. After these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY or pS and pT via diverse mechanisms related to cell shape, thereby affecting RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.

Resistance mechanism to Notch inhibition and combination therapy in human T-cell acute lymphoblastic leukemia.

Gain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T-cell acute lymphoblastic leukemia (T-ALL), highlighting the Notch signaling pathway as a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PI3K/AKT signaling, which regulates cell cycle and the spliceosome machinery, both at the transcriptional and posttranslational level. Moreover, several therapeutic combinations have been identified, in which simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models.

Proteome adaptations of the organohalide-respiring Desulfitobacterium hafniense strain DCB-2 to various energy metabolisms.

Introduction: Desulfitobacterium hafniense was isolated for its ability to use organohalogens as terminal electron acceptors via organohalide respiration (OHR). In contrast to obligate OHR bacteria, Desulfitobacterium spp. show a highly versatile energy metabolism with the capacity to use different electron donors and acceptors and to grow fermentatively. Desulfitobacterium genomes display numerous and apparently redundant members of redox enzyme families which confirm their metabolic potential. Nonetheless, the enzymes responsible for many metabolic traits are not yet identified. Methods: In the present work, we conducted an extended proteomic study by comparing the proteomes of Desulfitobacterium hafniense strain DCB-2 cultivated in combinations of electron donors and acceptors, triggering five alternative respiratory metabolisms that include OHR, as well as fermentation. Tandem Mass Tag labelling proteomics allowed us to identify and quantify almost 60% of the predicted proteome of strain DCB-2 (2,796 proteins) in all six growth conditions. Raw data are available via ProteomeXchange with identifier PXD030393. Results and discussion: This dataset was analyzed in order to highlight the proteins that were significantly up-regulated in one or a subset of growth conditions and to identify possible key players in the different energy metabolisms. The addition of sodium sulfide as reducing agent in the medium - a very widespread practice in the cultivation of strictly anaerobic bacteria - triggered the expression of the dissimilatory sulfite reduction pathway in relatively less favorable conditions such as fermentative growth on pyruvate, respiration with H2 as electron donor and OHR conditions. The presence of H2, CO2 and acetate in the medium induced several metabolic pathways involved in carbon metabolism including the Wood-Ljungdahl pathway and two pathways related to the fermentation of butyrate that rely on electron-bifurcating enzymes. While the predicted fumarate reductase appears to be constitutively expressed, a new lactate dehydrogenase and lactate transporters were identified. Finally, the OHR metabolism with 3-chloro-4-hydroxyphenylacetate as electron acceptor strongly induced proteins encoded in several reductive dehalogenase gene clusters, as well as four new proteins related to corrinoid metabolism. We believe that this extended proteomic database represents a new landmark in understanding the metabolic versatility of Desulfitobacterium spp. and provides a solid basis for addressing future research questions.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region.

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

Function-guided proximity mapping unveils electrophilic-metabolite sensing by proteins not present in their canonical locales.

Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs-despite their direct relevance to covalent drug development-impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs. In a setup that contrasts nucleus- vs. cytoplasm-specific responsivity to reactive-metabolite modification (hydroxynonenylation), ∼40% of the top-enriched protein sensors investigated respond in compartments of nonprimary origin or where the canonical activity of the protein sensor is inoperative. CDK9-a primarily nuclear-localized kinase-was hydroxynonenylated only in the cytoplasm. Site-specific CDK9 hydroxynonenylation-which we identified in untreated cells-drives its nuclear translocation, downregulating RNA-polymerase-II activity, through a mechanism distinct from that of commonly used CDK9 inhibitors. Taken together, this work documents an unmet approach to quantitatively profile and decode localized and context-specific signaling/signal-propagation programs orchestrated by reactive covalent ligands.

Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine.

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

Lysozyme-Induced Transcriptional Regulation of TNF-α Pathway Genes in Cells of the Monocyte Lineage.

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-α/IL-1ß pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA® identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles.

Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity.

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.

Tumor-associated factors are enriched in lymphatic exudate compared to plasma in metastatic melanoma patients.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137+ and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.

Analysis of the S. pombe Meiotic Proteome Reveals a Switch from Anabolic to Catabolic Processes and Extensive Post-transcriptional Regulation.

Meiotic progression in S. pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts, and targeted proteolysis of regulatory proteins. We have used SILAC labeling to examine the relative levels of proteins in diploid S. pombe cells during meiosis. Among the 3,268 proteins quantified at all time points, the levels of 880 proteins changed at least 2-fold; the majority of proteins showed stepwise increases or decreases during the meiotic divisions, while some changed transiently. Overall, we observed reductions in proteins involved in anabolism and increases in proteins involved in catabolism. We also observed increases in the levels of proteins of the ESCRT-III complex and revealed a role for ESCRT-III components in chromosome segregation and spore formation. Correlation with studies of meiotic gene expression and ribosome occupancy reveals that many of the changes in steady-state protein levels are post-transcriptional.

The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC.

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR.

Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis.

Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components.

Primary Human and Rat ß-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity.

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular ß-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by ß-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1.

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite's life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.

A mammalian transcription factor-specific peptide repository for targeted proteomics.

Site-specific transcription factors (TFs) play an essential role in mammalian development and function as they are vital for the majority of cellular processes. Despite their biological importance, TF proteomic data is scarce in the literature, likely due to difficulties in detecting peptides as the abundance of TFs in cells tends to be low. In recent years, significant improvements in MS-based technologies in terms of sensitivity and specificity have increased the interest in developing quantitative methodologies specifically targeting relatively lowly abundant proteins such as TFs in mammalian models. Such efforts would be greatly aided by the availability of TF peptide-specific information as such data would not only enable improvements in speed and accuracy of protein identifications, but also ameliorate cross-comparisons of quantitative proteomics data and allow for a more efficient development of targeted proteomics assays. However, to date, no comprehensive TF proteotypic peptide database has been developed. To address this evident lack of TF peptide data in public repositories, we are generating a comprehensive, experimentally derived TF proteotypic peptide spectral library dataset based on in vitro protein expression. Our library currently contains peptide information for 89 TFs and this number is set to increase in the near future. All MS data have been deposited in the ProteomeXchange with identifier PXD001212 (http://proteomecentral.proteomexchange.org/dataset/PXD001212).

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.