Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not “heat-killed” fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption–ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells

Quantitative proteomics analysis of CaMKII phosphorylation and the CaMKII interactome in the mouse forebrain

Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity and modulates subcellular targeting and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca(2+)-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKIIα and CaMKIIβ. CaMKII holoenzymes were then immunoprecipitated from subcellular fractions of forebrains isolated from either wild-type (WT) mice or mice with a Thr286 to Ala knock-in mutation of CaMKIIα (T286A-KI mice) and analyzed using the same approach in order to characterize in vivo phosphorylation sites in both CaMKII isoforms and identify CaMKII-associated proteins (CaMKAPs). A total of six and seven autophosphorylation sites in CaMKIIα and CaMKIIβ, respectively, were detected in WT mice. Thr286-phosphorylated CaMKIIα and Thr287-phosphorylated CaMKIIβ were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKIIα and Ser315- and Thr320/Thr321-phosphorylated CaMKIIβ were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKIIα at Ser275 across all subcellular fractions and of cytosolic CaMKIIβ at Ser315 and Thr320/Thr321. Significantly more CaMKAPs coprecipitated with WT CaMKII holoenzymes in the synaptic fraction compared to that in the membrane fraction, with functions including scaffolding, microtubule organization, actin organization, ribosomal function, vesicle trafficking, and others. The T286A-KI mutation altered the interactions of multiple CaMKAPs with CaMKII, including several proteins linked to autism spectrum disorders. These data identify CaMKII isoform phosphorylation sites and a network of synaptic protein interactions that are sensitive to the abrogation of Thr286 autophosphorylation of CaMKIIα, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice

Critical role of SIK3 in mediating high salt and IL-17 synergy leading to breast cancer cell proliferation

Chronic inflammation is a well-known precursor for cancer development and proliferation. We have recently demonstrated that high salt (NaCl) synergizes with sub-effective interleukin (IL)-17 to induce breast cancer cell proliferation. However, the exact molecular mechanisms mediating this effect are unclear. In our current study, we adopted a phosphoproteomic-based approach to identify salt modulated kinase-proteome specific molecular targets. The phosphoprotemics based binary comparison between heavy labelled MCF-7 cells treated with high salt (Δ0.05 M NaCl) and light labelled MCF-7 cells cultured under basal conditions demonstrated an enhanced phosphorylation of Serine-493 of SIK3 protein. The mRNA transcript and protein expression analysis of SIK3 in MCF-7 cells demonstrated a synergistic enhancement following co-treatment with high salt and sub-effective IL-17 (0.1 ng/mL), as compared to either treatments alone. A similar increase in SIK3 expression was observed in other breast cancer cell lines, MDA-MB-231, BT20, and AU565, while non-malignant breast epithelial cell line, MCF10A, did not induce SIK3 expression under similar conditions. Biochemical studies revealed mTORC2 acted as upstream mediator of SIK3 phosphorylation. Importantly, cell cycle analysis by flow cytometry demonstrated SIK3 induced G0/G1-phase release mediated cell proliferation, while SIK3 silencing abolished this effect. Also, SIK3 induced pro-inflammatory arginine metabolism, as evidenced by upregulation of the enzymes iNOS and ASS-1, along with downregulation of anti-inflammatory enzymes, arginase-1 and ornithine decarboxylase. Furthermore, gelatin zymography analysis has demonstrated that SIK3 induced expression of tumor metastatic CXCR4 through MMP-9 activation. Taken together, our data suggests a critical role of SIK3 in mediating three important hallmarks of cancer namely, cell proliferation, inflammation and metastasis. These studies provide a mechanistic basis for the future utilization of SIK3 as a key drug discovery target to improve breast cancer therapy

Differential Localization of G Protein βγ Subunits

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system

Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction

Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD

Imaging Cataract-Specific Peptides in Human Lenses

Age-related protein truncation is a common process in long-lived proteins such as proteins found in the ocular lens. Major truncation products have been reported for soluble and membrane proteins of the lens, including small peptides that can accelerate protein aggregation. However, the spatial localization of age-related protein fragments in the lens has received only limited study. Imaging mass spectrometry (IMS) is an ideal tool for examining the spatial localization of protein products in tissues. In this study we used IMS to determine the spatial localization of small crystallin fragments in aged and cataractous lenses. Consistent with previous reports, the pro-aggregatory αA-crystallin 66–80 peptide as well as αA-crystallin 67–80 and γS-crystallin 167–178 were detected in normal lenses, but found to be increased in nuclear cataract regions. In addition, a series of γS-crystallin C-terminal peptides were observed to be mainly localized to cataractous regions and barely detected in transparent lenses. Other peptides, including abundant αA3-crystallin peptides were present in both normal and cataract lenses. The functional properties of these crystallin peptides remain unstudied; however, their cataract-specific localization suggests further studies are warranted

Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS

Tutkimuksen tarkoituksena on selvittää, onko kaksikielisten lasten eri kielten sanastojen monipuolisuudessa eroavaisuuksia. Tätä lähden tutkimaan vertailemalla substantiivien ja verbien sanemääriä eri kielillä, selvittämällä sanastojen toisteisuutta sekä tutkimalla sanatoisteisuuden ja aineistossa kerran esiintyvien sanojen välistä riippuvuutta. Lisäksi haluan selvittää, onko sukupuolten välillä eroavaisuuksia sanaston monipuolisuutta tarkastellessa. Tutkimuskysymyksiä syntyi kaksi. Eroaako kaksikielisten lasten eri kielten sanastojen monipuolisuus toisistaan, ja löytyykö sukupuolten väliltä eroavaisuuksia sanastojen monipuolisuuden osalta? Ensimmäiseen tutkimuskysymykseen vastaan kolmella alakysymyksellä. Eroavatko lasten erikieliset tarinat sanemääriltään toisistaan, onko sanasto toisteisempaa toisella kielellä kuin toisella, ja onko sanojen toisteisuuden ja aineistossa kerran esiintyvien sanojen välillä yhteys? Tavoitteena on saada lisää tietoa kaksikielisten lasten sanaston monipuolisuudesta narratiivisella tutkimusmenetelmällä. On tärkeää saada tietoa kaksikielisten lasten sanaston monipuolisuudesta, sillä kaksi- ja monikielisten perheiden määrä on Suomessa koko ajan lisääntymässä. Tutkimus on monimenetelmällinen tutkimus, sillä aineisto kerättiin laadullisin menetelmin, mutta analysoitiin määrällisesti. Aineisto kerättiin sadutusmenetelmällä kevään ja syksyn aikana 2019. Tutkimushenkilöitä oli kahdeksan. Kahdeksan neljä-viisivuotiasta suomalaisranskalaista lasta, joiden toisen vanhemman äidinkieli on suomi ja toisen ranska. Tutkimushenkilöiden valinnan kriteerinä oli myös se, että lapsi asuu Suomessa ja käy ranskankielistä päiväkotia. Aineiston analyysissä käytettiin TTR-arvoa ja hapax legomenon -lekseemejä. Tämän lisäksi TTR-arvojen ja HL-lekseemien välille laskettiin korrelaatio, jotta voidaan nähdä, onko sanojen toisteisuuden ja aineistossa kerran esiintyvien sanojen välillä yhteys. Tutkimustuloksissa ilmeni, että suomen kielen sanasto on lapsilla monipuolisempaa kuin ranskan kielen sanasto. Sukupuolten välillä ei ilmennyt huomattavia eroavaisuuksia sanastojen monipuolisuuden osalta. Eri mittareilla saatujen tulosten välillä todettiin positiivinen korrelaatio molemmilla kielillä. Tämä tutkimus on kuvaileva tutkimus, joten tutkimustuloksia ei ole tarkoitus yleistää

Mapping glycation and glycoxidation sites in collagen I of human cortical bone

Accumulation of advanced glycation end products (AGEs), particularly in long-lived extracellular matrix proteins, has been implicated in pathogenesis of diabetic complications and in aging. Knowledge about specific locations of AGEs and their precursors within protein primary structure is critical for understanding their physiological and pathophysiological impact. However, the information on specific AGE sites is lacking. Here, we identified sequence positions of four major AGEs, carboxymethyllysine, carboxyethyllysine, 5-hydro-5-methyl imidazolone, and 5-hydro-imidazolone, and an AGE precursor fructosyllysine within the triple helical region of collagen I from cortical bone of human femurs. The presented map provides a basis for site-specific quantitation of AGEs and other non-enzymatic post-translational modifications and identification of those sites affected by aging, diabetes, and other diseases such as osteoporosis; it can also help in guiding future studies of AGE impact on structure and function of collagen I in bone

Spatially-Directed Protein Identification from Tissue Sections by Top-Down LC-MS/MS with Electron Transfer Dissociation

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 μL extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50–100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations

Mass Spectrometric Methods for the Analysis of Nucleoside–Protein Cross-Links: Application to Oxopropenyl-deoxyadenosine

Electrophilic DNA adducts produced following oxidative stress can form DNA–protein cross-links (DPCs), dramatically altering genomic maintenance pathways. Complete characterization of DPCs has been hindered, in part, because of a lack of comprehensive techniques for their analysis. We have, therefore, established a proteomics approach to investigate sites of cross-link formation using <i>N</i>^<i>6</i>-(3-oxo-1-propenyl)-2′-deoxyadenosine (OPdA), an electrophilic DNA adduct produced from oxidative stress. OPdA was reacted with albumin and reduced with NaBH₄ to stabilize DPCs. Using LC-MS/MS proteomics techniques, high-resolution peptide sequence data were obtained; however, using a database searching strategy, adducted peptides were only identified in samples subjected to chemical depurination. This strategy revealed multiple oxopropenyl adenine-lysine adducts and oxopropenyl-lysine adducts with the most reactive lysines identified to be Lys256 and Lys548. Manual interrogation of the mass spectral data provided evidence of OPdA deoxynucleoside conjugates to lysines and cross-links that underwent facile collision-induced dissociation to release an unmodified peptide without subsequent fragmentation. These fragmentations precluded adduct detection and peptide sequencing using database searching methods. Thus, comprehensive analysis of DPCs requires chemical depurination of DNA–protein reaction mixtures followed by a combination of database-dependent and manual interrogation of LC-MS/MS data using higher-energy collision-induced dissociation. In the present case, this approach revealed that OPdA selectively modifies surface lysine residues and produces nucleoside–protein cross-links and oxopropenyl lysine