Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two dimensional electrophoresis and nano-electrospray ionization ion trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (pI 4.8) as isoforms of β-tubulin. This identification was confirmed by immuno-blot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI). A 6-AziP adduct of TAVCDIPPR (m/z=1287.77), a β-tubulin specific peptide, was detected by MALDI. High resolution LC-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.
tubulin; mass spectrometry; photolabeling; neurosteroids
Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin (Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts.
We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/µg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates eight Ser/Thr residues in Cx45 in vitro.
Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.
connexin45; gap junctions; quantitative immunoblots; co-immunoprecipitation; electron microscopy; Cx45 phosphorylation; CaMKII; transgenic mice
The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. While its activation of p53 has been well characterized, ARF’s role in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5 which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, since reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis.
ARF; DDX5; NPM; non-oncogene; ribosome biogenesis
The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (IA) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting and cell surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na+ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases IA densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of heterologously expressed Kv4.2 protein, resulting in greater total and cell surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Taken together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded IA channel complexes and a novel regulator of IA channel densities and neuronal excitability.
The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.
The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.
Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
Ischemia repurfusion injury; Two dimensional gel electrophoresis; Mass spectrometry; Liver transplantation; Chaperonins; Endoplasmic reticulum (ER) stress
Voltage-gated K+ (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as IA (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as Ito,f (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal IA and cardiac Ito,f channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional IA and Ito,f channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and, importantly, unbiased, approach to identify the components of native macromolecular protein complexes. The recent application of proteomic approaches to identify the components of native neuronal (and cardiac) Kv4 channel complexes has revealed even greater complexity than anticipated. The continued emphasis on development of improved biochemical and analytical proteomics methods seems certain to accelerate progress and to provide important new insights into the molecular determinants of native ion channel protein complexes.
Proteomics; Protein identification; Native ion channel protein complexes; Kv4 α subunits; Kv accessory subunits; Post-translational modifications; Quantitative mass spectrometry
Using a proteomics approach, the authors examined whether class 1 UV-blocking contact lenses protect against UVB radiation–induced damage in a human lens epithelial cell line (HLE B-3) and postmortem human lenses.
To determine whether class 1 UV-blocking contact lenses protect against UVB radiation–induced damage in a human lens epithelial cell line (HLE B-3) and postmortem human lenses using a proteomics approach.
HLE B-3 cells were exposed to 6.4 mW/cm2 UVB radiation at 302 nm for 2 minutes (768 mJ/cm2) with or without covering by senofilcon A class 1 UV-blocking contact lenses or lotrafilcon A non–UV-blocking (lotrafilcon A has some UV-blocking ability, albeit minimal) contact lenses. Control cells were not exposed to UVB radiation. Four hours after treatment, cells were analyzed by two-dimensional difference gel electrophoresis and tandem mass spectrometry, and changes in protein abundance were quantified. F-actin and microtubule cytoskeletons were examined by fluorescence staining. In addition, human donor lenses were exposed to UVB radiation at 302 nm for 4 minutes (1536 mJ/cm2). Cortical and epithelial cell proteins were scraped from lens surfaces and subjected to the same protein analyses.
Senofilcon A lenses were beneficial for protecting HLE B-3 cells against UVB radiation–induced changes in caldesmon 1 isoform, lamin A/C transcript variant 1, DEAD (Asp-Glu-Ala-Asp) box polypeptide, β-actin, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), annexin A2, triose phosphate isomerase, and ubiquitin B precursor. These contact lenses also prevented actin and microtubule cytoskeleton changes typically induced by UVB radiation. Conversely, non–UV-blocking contact lenses were not protective. UVB-irradiated human lenses showed marked reductions in αA-crystallin, αB-crystallin, aldehyde dehydrogenase 1, βS-crystallin, βB2-crystallin, and G3PDH, and UV-absorbing contact lenses significantly prevented these alterations.
Senofilcon A class 1 UV-blocking contact lenses largely prevented UVB-induced changes in protein abundance in lens epithelial cells and in human lenses.
Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multi-functional protein kinase, Ca2+/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca2+ homeostasis, transcription, apoptosis and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO2, and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these “orthogonal” methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.
serine phosphorylation; Orbitrap; ETD; CID; C-terminal fusion protein
Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.
Nucleophosmin (NPM/B23) is a multifunctional oncoprotein whose protein expression levels dictate cellular growth and proliferation rates. NPM is translationally responsive to hyperactive mammalian target of rapamycin (mTOR) signals, but the mechanism of this regulation is not understood. Using chimeric translational reporters, we found that the 3’ untranslated region (UTR) of the NPM mRNA is sufficient to mediate its translational modulation by mTOR signalling. We show that far upstream element (FUSE)-binding protein 1 (FBP1) interacts specifically with the 3’ UTR of NPM to repress translation. Overexpression of FBP1 resulted in translational repression of NPM mRNAs, while depletion of FBP1 caused a dramatic increase in NPM translation and resulted in enhanced overall cell proliferation. Thus, we propose that FBP1 is a key regulator of cell growth and proliferation through its ability to selectively bind the NPM 3’ UTR and repress NPM translation.
FBP1; Nucleophosmin; Ribosome Biogenesis; Translation
Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 γ-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the α1 subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α1 subunit was observed in all three replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 γ-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the α1 subunit, confirmed two splice variants of the γ2 subunit (γ2L and γ2S) and resolved a database discrepancy in the sequence of the α5 subunit.
Biliary atresia (BA) is the most serious liver disease in infants. Diagnosis currently depends on surgical exploration of the biliary tree. Non-invasive tests that distinguish BA from other types of neonatal liver disease are not available.
To identify potential serum biomarkers that classify children with neonatal cholestasis, we performed 2-dimensional difference gel electrophoresis, statistical analysis, and tandem mass spectrometry using serum samples from 19 infants with BA and 19 infants with non-BA neonatal cholestasis.
11 potential serum biomarkers were found that could in combination classify children with neonatal cholestasis.
Although no single biomarker or imaging test adequately distinguishes BA from other types of neonatal cholestasis, combinations of biomarkers, imaging tests and non-invasive clinical criteria should be further explored as potential tests for rapid and accurate diagnosis of BA.
Proteomics; biliary atresia; neonatal cholestasis; biomarker
Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via two-dimensional difference gel electrophoresis (2-D DIGE) that were expressed at either higher or lower levels (>3-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the endoplasmic reticulum stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.
2-D DIGE; biomarkers; cervical cancer; cDNA microarray; RNAlater
The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.
Decoration of proteins with carbohydrates has an important impact on protein function throughout biology and has been recognized increasingly in pathogenic bacteria. Haemophilus influenzae is a common cause of both bacterial respiratory tract disease and bacterial invasive disease and initiates infection by colonizing the upper respiratory tract. The Haemophilus HMW1 adhesin is a large protein that resides on the bacterial surface and mediates bacterial attachment to respiratory epithelial cells, an essential step in the process of colonization. In recent work, we discovered that HMW1 is decorated at multiple sites with short carbohydrate units that serve to prevent degradation and to stabilize association with the bacterial surface. In the current study we identify the enzyme responsible for adding carbohydrate units at specific sites of HMW1. In addition, we demonstrate that this enzyme is capable of creating both carbohydrate-protein and carbohydrate-carbohydrate bonds. The amino acid sequence of this enzyme is similar to the sequences of proteins in several other bacteria, suggesting a new family of bacterial enzymes capable of creating carbohydrate-protein and carbohydrate-carbohydrate bonds.
While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of >24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.
Anoxia; proteomic; zebrafish
Somatodendritic A-type (IA) voltage-gated K+ (KV) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic IA channels are encoded by KV4 α subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain KV4.2-encoded IA channel complexes. Using anti-KV4.2 specific antibodies, KV4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (KV4.2−/−) mice harboring a targeted disruption of the KCND2 (KV4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D-or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the KV4 α subunits (KV4.2, KV4.3 and KV4.1) and the KV4 accessory, KChIP (KChIPI-4) and DPP (DPP6 and 10), proteins in native brain KV4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the KV channel accessory subunit KVβ1, are also components of native brain KV4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified KV4 interacting proteins.
IA; accessory subunits; mass spectrometric identification
Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.
The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1∶IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1∶IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1∶IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.
Infection with Entamoeba histolytica can result in disabling diarrhea or even death, while the morphologically identical and genetically similar Entamoeba dispar harmlessly colonizes the human intestine. Understanding the molecular differences between these two organisms by comparing their protein repertoire may help us to understand why E. histolytica invades into colonic tissue, while E. dispar remains a benign passenger. Here, we identify four proteins that appear to be differentially expressed between the two species and show that a metabolic enzyme, which would appear to be an unlikely candidate for a role in disease, is expressed at much higher levels in the pathogenic organism.
Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by the administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for β cell function, suggesting a vital framework for the regulation of glucose homeostasis.
DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579–586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12−/− mice to septic shock. We show that DAP12−/− mice have improved survival from both endotoxemia and cecal ligation and puncture–induced septic shock. As compared with WT mice, DAP12−/− mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.
Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.
Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics.
Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5%) whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and individual samples.
Quantitative label-free LC-MS/MS can measure scores of CSF proteins with low technical variability and can segregate samples according to desired criteria. Thus, this technique shows potential for biomarker discovery for neurological diseases.
Disease-modifying therapies for Alzheimer’s disease (AD) would be most beneficial if applied during the ‘preclinical’ stage (pathology present with cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that can detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design.
2D–difference gel electrophoresis and liquid chromatography tandem mass spectrometry were used to measure AD-associated changes in cerebrospinal fluid (CSF). Concentrations of CSF YKL-40 were further evaluated by enzyme-linked immunosorbent assay in the discovery cohort (N=47), an independent sample set (N=292) with paired plasma samples (N=237), frontotemporal lobar degeneration (N=9), and progressive supranuclear palsy (PSP, N=6). Human AD brain was studied immunohistochemically to identify potential source(s) of YKL-40.
In the discovery and validation cohorts, mean CSF YKL-40 was higher in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) vs. controls (CDR 0) and PSP. Importantly, CSF YKL-40/Aβ42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) as well as the best CSF biomarkers identified to date, tau/Aβ42 and p-tau181/Aβ42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 vs. CDR 0 groups, and correlated with CSF levels. YKL-40 immunoreactivity was observed within astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aβ deposition.
These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD, and that, together with Aβ42, has potential prognostic utility as a biomarker for preclinical AD.
YKL-40; Alzheimer’s disease; biomarkers; cerebrospinal fluid; chitinase-3 like-1; inflammation
The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown.
Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations.
A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis.
DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.)
Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the ‘preclinical’ stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.
Methods and Findings
CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85–0.94 95% confidence interval [CI]) and 0.88 (0.81–0.94 CI), respectively.
Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.