Amot130 adapts atrophin-1 interacting protein 4 to inhibit yes-associated protein signaling and cell growth.

The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.

Identification, analysis, and prediction of protein ubiquitination sites.

Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry, and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory, and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associatedmutations. UbPred is available at http://www.ubpred.org.

Tumor Necrosis Factor-alpha- and interleukin-1-induced cellular responses: coupling proteomic and genomic information.

The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFalpha) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFalpha- and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFalpha and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFalpha and IL-1 regulate different processes. A large-scale proteomic analysis of TNFalpha- and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFalpha and IL-1. When combined with genomic studies, our results indicate that TNFalpha, but not IL-1, mediates cell cycle arrest.

Comparative proteomic analysis of human CD34+ stem/progenitor cells and mature CD15+ myeloid cells.

Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.

Proteomic analysis of differential protein expression induced by ultraviolet light radiation in HeLa cells.

Cells treated with ultraviolet (UV) radiation undergo cell cycle arrest at the S-phase and G1/S boundary, allowing DNA repair to occur. Several proteins such as replication protein A and DNA-dependent protein kinase have been suggested to be involved in UV-induced inhibition of DNA replication. However, the role of these proteins in inhibiting DNA replication remains unknown. Other proteins may play important roles in modulating functions of these proteins in response to UV-irradiation. To understand the broad range of proteins involved in this inhibition, we carried out a systematic study to identify specific proteins involved in UV-induced replication arrest using two-dimensional gel electrophoresis and mass spectrometry. Unique changes in protein expression level for 31 proteins were observed over a 24-hour time course, including calgizzarin, cyclophilin A, and macrophage migration inhibitory factor. The expression level changes of these proteins are dynamically correlated to DNA replication activity, suggesting involvement of these proteins in modulating DNA replication and repair activities. This proteomic approach provides opportunities to gain insights into the mechanism by which DNA replication is inhibited.

Rapid determination of advanced glycation end products of proteins using MALDI-TOF-MS and PERL script peptide searching algorithm.

Advanced glycation end products (AGEs), which are composed of various glucose or carbohydrate adducts, are thought to be responsible for several diabetic and age-related complications. However, to date, specific sites on proteins that are modified by AGEs remain largely unknown. We report here the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine the type and localization of several AGEs formed in vitro on human beta-2-microglobulin (beta2M), and in vivo on type 2 ryanodine receptor calcium-release channel (RyR2), and sarco(endo)plasmic reticulum (SERCA2a). A PERL script algorithm, developed in-house, makes searching the relatively large amount of data generated by the MALDI-MS more manageable. The outstanding sensitivity of MALDI-TOF-MS coupled with the PERL script algorithm allows such an approach to be a very useful tool in detecting AGEs and other post-translational modifications. We believe that this method could be an important tool when searching for post-translational modifications on proteins.

Identification of methylation and acetylation sites on mouse histone H3 using matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization tandem mass spectrometry.

Covalent modifications to histone proteins are well documented in the literature. Specific modification sites are correlated with chromatin structure and transcriptional activity. The histone code is very complex, and includes several types of covalent modifications such as acetylation, methylation, phosphorylation, and ubiquitination of at least 20 possible sites within the histone proteins. The final chromatin structure "read-out" is a result of the cooperation between these many sites of covalent modifications. Methylation and acetylation sites of histone H3 from many different species have been previously identified. However, a full post-translational modification status on histone H3 from mouse has not yet been reported. Here we demonstrate the use of high-accuracy matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization tandem mass spectrometry to identify the methylation and acetylation sites of the mouse histone H3. In addition to the sites previously identified from other species, one unique methylation site, Lys-122, from mouse histone H3 was identified. The reported mass spectrometric method provides an efficient and sensitive way for analyzing post-translational modifications of histone proteins.

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.

Chronic diabetes increases advanced glycation end products on cardiac ryanodine receptors/calcium-release channels.

Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H(+)) >or=500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H(+) peaks corresponding to theoretical RyR2 peptides with single N(epsilon)-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction.