PFN4 is required for manchette development and acrosome biogenesis during mouse spermiogenesis.

Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.

Insights into the Mechanism of Human Deiodinase 1.

The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.

Proteomic analysis demonstrates the role of the quality control protease LONP1 in mitochondrial protein aggregation.

The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with aging. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells showed a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by a reduced LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human aging processes.

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice.

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Quantitative proteomics of synaptosome S-nitrosylation in Alzheimer's disease.

Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.

Evolutionary redesign of the lysosomal enzyme arylsulfatase A increases efficacy of enzyme replacement therapy for metachromatic leukodystrophy.

Protein engineering is a means to optimize protein therapeutics developed for the treatment of so far incurable diseases including cancers and genetic disorders. Here we report on an engineering approach in which we successfully increased the catalytic rate constant of an enzyme that is presently evaluated in enzyme replacement therapies (ERT) of a lysosomal storage disease (LSD). Although ERT is a treatment option for many LSDs, outcomes are lagging far behind expectations for most of them. This has been ascribed to insufficient enzyme activities accumulating in tissues difficult to target such as brain and peripheral nerves. We show for human arylsulfatase A (hARSA) that the activity of a therapeutic enzyme can be substantially increased by reversing activity-diminishing and by inserting activity-promoting amino acid substitutions that had occurred in the evolution of hominids and non-human mammals, respectively. The potential of this approach, here designated as evolutionary redesign, was highlighted by the observation that murinization of only 1 or 3 amino acid positions increased the hARSA activity 3- and 5-fold, with little impact on stability, respectively. The two kinetically optimized hARSA variants showed no immunogenic potential in ERT of a humanized ARSA knockout mouse model of metachromatic leukodystrophy (MLD) and reduced lysosomal storage of kidney, peripheral and central nervous system up to 3-fold more efficiently than wild-type hARSA. Due to their safety profile and higher therapeutic potential the engineered hARSA variants might represent major advances for future enzyme-based therapies of MLD and stimulate analogous approaches for other enzyme therapeutics.

Analysis of heat-induced protein aggregation in human mitochondria.

Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.

IMiQ: a novel protein quality control compartment protecting mitochondrial functional integrity.

Aggregation processes can cause severe perturbations of cellular homeostasis and are frequently associated with diseases. We performed a comprehensive analysis of mitochondrial quality and function in the presence of aggregation-prone polypeptides. Despite a significant aggregate formation inside mitochondria, we observed only a minor impairment of mitochondrial function. Detoxification of aggregated reporter polypeptides as well as misfolded endogenous proteins inside mitochondria takes place via their sequestration into a specific organellar deposit site we termed intramitochondrial protein quality control compartment (IMiQ). Only minor amounts of endogenous proteins coaggregated with IMiQ deposits and neither resolubilization nor degradation by the mitochondrial protein quality control system were observed. The single IMiQ aggregate deposit was not transferred to daughter cells during cell division. Detoxification of aggregates via IMiQ formation was highly dependent on a functional mitochondrial fission machinery. We conclude that the formation of an aggregate deposit is an important mechanism to maintain full functionality of mitochondria under proteotoxic stress conditions.

Distinct Expression and Function of FcεRII in Human B Cells and Monocytes.

FcεRII is a multifunctional low-affinity IgER that is involved in the pathogenesis of allergic, inflammatory, and neoplastic diseases. Although discrepancies in FcεRII-mediated functions are being increasingly recognized, the consequences of FcεRII activation are not completely understood. In this study, we evaluated the expression of FcεRII on human blood cells and found that it was primarily expressed on monocytes and B cells. Although IL-4 promoted expression of the FcεRIIb isoform on B cells and monocytes, the expression of the FcεRIIa isoform was not dependent on IL-4. Furthermore, FcεRII predominantly bound allergen-IgE complexes on B cells but not on monocytes. FcεRII-mediated allergen-IgE complex uptake by B cells directed Ags to MHC class II-rich compartments. FcεRII-bearing monocytes and B cells expressed high levels of the FcεRII sheddase a disintegrin and metalloproteinase 10, which implies that they are important sources of soluble FcεRII. Moreover, we identified that IgE immune complex stimulation of FcεRII activated intracellular tyrosine phosphorylation via Syk in B cells but not in monocytes. Importantly, FcεRII-mediated signaling by allergen-IgE immune complexes increased IFN-γ production in B cells of allergic patients during the build-up phase of allergen-specific immunotherapy. Together, our results demonstrate that FcεRII mediates cell type-dependent function in allergic reactions. In addition, the results identify a novel allergen-IgE complex/FcεRII/Syk/IFN-γ pathway in allergic responses and suggest that FcεRII may play a role in regulating allergic reactions via modulating IFN-γ production in B cells.

Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris.

CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.

Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics.

Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed RAW 264.7 macrophages to C. albicans for 3h and used SILAC to quantify macrophage proteins and phosphoproteins by mass spectrometry to study the effects of infection. We identified 53 macrophage up-regulated proteins and 15 less abundant in the presence of C. albicans out of a total of 2071 identified proteins. 922 unique protein phosphorylation sites were identified by phosphopeptide enrichment and mass spectrometry, including 327 previously unidentified mouse protein phosphorylation sites. 126 peptides showed an increase and 70 a decrease in their phosphorylation level. The majority of the differentially expressed and phosphorylated proteins are receptors, mitochondrial ribosomal proteins, cytoskeletal proteins, and transcription factor activators involved in inflammatory and oxidative responses. In addition, we identified 22 proteins and phosphoproteins related to apoptosis. The analysis of apoptotic markers revealed that anti-apoptotic signals prevailed during the interaction of the yeast. Our proteomics study suggests that besides inflammation, apoptosis is a central pathway in the immune defense against C. albicans infection. BIOLOGICAL SIGNIFICANCE: This work uses SILAC and SIMAC methodology combined with CPP (+ TiO2) to study protein and phosphopeptide changes in RAW 264.7 macrophages in response to coincubation with Candida albicans for 3h. We show that the presence of C. albicans induces inflammatory responses and inhibits apoptosis in the macrophages. Our phosphoproteomic analysis identified 327 new mouse protein phosphorylation sites.

Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry.

Reversible protein phosphorylation plays a critical role in cell signaling and is responsible for the regulation of many biological processes in most living organisms. The low stoichiometry of protein phosphorylation requires sensitive analysis by tandem mass spectrometry. However, incomplete peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho-site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ∼32 compared to ∼23 and ∼17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD), respectively. Compared with the Ascore using either CAD or ETD, the Cscore identified up to 88% more phosphorylation sites. Using a phosphopeptide library revealed that the score threshold for obtaining a false-localization rate of 0.5% was lower for the Cscore than either the Ascore (CAD) or the Ascore (ETD).

Time-resolved quantitative phosphoproteomics: new insights into Angiotensin-(1-7) signaling networks in human endothelial cells.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Our data suggest that the antiproliferative activity of Ang-(1-7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1-7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7). Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1-7) signal transduction, shedding light on the mechanisms underlying Mas activation.

Adhesion and degranulation promoting adapter protein (ADAP) is a central hub for phosphotyrosine-mediated interactions in T cells.

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

Redox-regulated conformational changes in an SH3 domain.

Oxidation-induced conformational changes in proteins provide a powerful mechanism to sense the redox state of a living cell. In contrast to the unspecific and often irreversible oxidation of intracellular proteins during severe oxidative stress, regulatory redox events need to have specific and transient effects on cellular targets. Here we present evidence for the reversible formation of a vicinal disulfide bond in a prototypic protein interaction domain. NMR spectroscopy was used to determine the structure of the N-terminal hSH3 domain (hSH3N) of the immune cell protein ADAP (adhesion and degranulation promoting adapter protein) in the reduced and oxidized states. An eight-membered ring formed upon oxidation of two neighboring cysteines leads to significant changes in the variable arginine-threonine (RT) loop of the hSH3N domain and alters the helix-sheet packing of the domain. The redox potential for this structural transition is -228 mV at pH 7.4. This is compatible with a role of the cysteinylcysteine moiety in redox signaling during T cell activation.