Non-nuclear Proteins Research Articles

Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain.

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.

Protein acetylation effects on enzyme activity and metabolic pathway fluxes

Acetylation of proteins seems a widespread process found in the three domains of life. Several studies have shown that besides histones, acetylation of lysine residues also occurs in non-nuclear proteins. Hence, it has been suggested that this covalent modification is a mechanism that might regulate diverse metabolic pathways by modulating enzyme activity, stability, and/or subcellular localization or interaction with other proteins. However, protein acetylation levels seem to have low correlation with modification of enzyme activity and pathway fluxes. In addition, the results obtained with mutant enzymes that presumably mimic acetylation have frequently been over-interpreted. Moreover, there is a generalized lack of rigorous enzyme kinetic analysis in parallel to acetylation level determinations. The purpose of this review is to analyze the current findings on the impact of acetylation on metabolic enzymes and its repercussion on metabolic pathways function/regulation.

The Role of Protein Acetylation in Centrosome Biology.

Acetylation is among the most prevalent posttranslational modifications in cells and regulates a number of physiological processes such as gene transcription, cell metabolism, and cell signaling. Although initially discovered on nuclear histones, many non-nuclear proteins have subsequently been found to be acetylated as well. The centrosome is the major microtubule-organizing center in most metazoans. Recent proteomic data indicate that a number of proteins in this subcellular compartment are acetylated. This review gives an overview of our current knowledge on protein acetylation at the centrosome and its functional relevance in organelle biology.

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins

ABSTRACTLaminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.

NLSdb-major update for database of nuclear localization signals and nuclear export signals.

NLSdb is a database collecting nuclear export signals (NES) and nuclear localization signals (NLS) along with experimentally annotated nuclear and non-nuclear proteins. NES and NLS are short sequence motifs related to protein transport out of and into the nucleus. The updated NLSdb now contains 2253 NLS and introduces 398 NES. The potential sets of novel NES and NLS have been generated by a simple ‘in silico mutagenesis’ protocol. We started with motifs annotated by experiments. In step 1, we increased specificity such that no known non-nuclear protein matched the refined motif. In step 2, we increased the sensitivity trying to match several different families with a motif. We then iterated over steps 1 and 2. The final set of 2253 NLS motifs matched 35% of 8421 experimentally verified nuclear proteins (up from 21% for the previous version) and none of 18 278 non-nuclear proteins. We updated the web interface providing multiple options to search protein sequences for NES and NLS motifs, and to evaluate your own signal sequences. NLSdb can be accessed via Rostlab services at: https://rostlab.org/services/nlsdb/

NLScore: a novel quantitative algorithm based on 3 dimensional structural determinants to predict the probability of nuclear localization in proteins containing classical nuclear localization signals.

The presence of a nuclear localization signal (NLS) in proteins can be inferred by the presence of a stretch of basic amino acids (KRKK). These NLSs are termed classical NLS (cNLS). However, only a fraction of proteins containing the cNLS pattern are transported into the nucleus by binding to importin α. Hence, there must exist, additional structural determinants that guide the appropriate interaction between putative NLSs containing cargo and importin α. Using 52 protein structures containing cNLS obtained from RCSB PDB, we assembled a training set and a validation set such that both sets were comprised of a combination of proteins with proven nuclear localization and ones that were non-nuclear. We modeled the interface between cargoes containing cNLS and importin α. We conducted rigid body docking and produced induced-fit modes by allowing both side chain and the backbone to be flexible. The output of these studies and additional determinants such as energy of interaction, atomic contacts, hydrophilic interaction, cationic interaction, and penetration of the cargo protein were used to derive a 26 parameter quantitative structure activity relationship based regression equation. This was further optimized by a step-wise backward elimination approach to derive a 15 parameter score. This NLScore was not only able to correctly classify confirmed nuclear and non-nuclear localized proteins but it was able to perform better than currently implemented algorithms like NucPred, Euk-mPLoc 2.0, cNls Mapper, and NLStradamus. Leave-one-out cross validation (LOOCV) showed that NLScore correctly predicted 78.6% and 81.6% of non-nuclear and nuclear proteins respectively. Graphical abstract NLScore: a novel quantitative algorithm based on 3 dimensional structural determinants to predict the probability of nuclear localization in proteins.

Autophagy-related intrinsically disordered proteins in intra-nuclear compartments.

Recent analyses indicated that autophagy can be regulated via some nuclear transcriptional networks and many important players in the autophagy and other forms of programmed cell death are known to be intrinsically disordered. To this end, we analyzed similarities and differences in the intrinsic disorder distribution of nuclear and non-nuclear proteins related to autophagy. We also looked at the peculiarities of the distribution of the intrinsically disordered autophagy-related proteins in various intra-nuclear organelles, such as the nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinucleolar compartment. This analysis revealed that the autophagy-related proteins constitute about 2.5% of the non-nuclear proteins and 3.3% of the nuclear proteins, which corresponds to a substantial enrichment by about 32% in the nucleus. Curiously, although, in general, the autophagy-related proteins share similar characteristics of disorder with a generic set of all non-nuclear proteins, chromatin and nuclear speckles are enriched in the intrinsically disordered autophagy proteins (29 and 37% of these proteins are disordered, respectively) and have high disorder content at 0.24 and 0.27, respectively. Therefore, our data suggest that some of the nuclear disordered proteins may play important roles in autophagy.

Compartmentalization and Functionality of Nuclear Disorder: Intrinsic Disorder and Protein-Protein Interactions in Intra-Nuclear Compartments.

The cell nucleus contains a number of membrane-less organelles or intra-nuclear compartments. These compartments are dynamic structures representing liquid-droplet phases which are only slightly denser than the bulk intra-nuclear fluid. They possess different functions, have diverse morphologies, and are typically composed of RNA (or, in some cases, DNA) and proteins. We analyzed 3005 mouse proteins localized in specific intra-nuclear organelles, such as nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinuclear compartment and compared them with ~29,863 non-nuclear proteins from mouse proteome. Our analysis revealed that intrinsic disorder is enriched in the majority of intra-nuclear compartments, except for the nuclear pore and lamina. These compartments are depleted in proteins that lack disordered domains and enriched in proteins that have multiple disordered domains. Moonlighting proteins found in multiple intra-nuclear compartments are more likely to have multiple disordered domains. Protein-protein interaction networks in the intra-nuclear compartments are denser and include more hubs compared to the non-nuclear proteins. Hubs in the intra-nuclear compartments (except for the nuclear pore) are enriched in disorder compared with non-nuclear hubs and non-nuclear proteins. Therefore, our work provides support to the idea of the functional importance of intrinsic disorder in the cell nucleus and shows that many proteins associated with sub-nuclear organelles in nuclei of mouse cells are enriched in disorder. This high level of disorder in the mouse nuclear proteins defines their ability to serve as very promiscuous binders, possessing both large quantities of potential disorder-based interaction sites and the ability of a single such site to be involved in a large number of interactions.

Genetic risk alleles associated with serologic autoimmunity in normal individuals (BA3P.113)

Abstract We screened 2628 normal individuals for antibodies binding to the mix of antigens in a human nuclear extract and identified 579 (26.05%) individuals with significant levels of IgG antibodies to self-antigens. Next, we analyzed 639 representative sera from ANA-, ANA+, ILE and SLEs on an autoantigen proteomic array, which identified 33 antigens strongly recognized by IgG autoantibodies in ANA+ group as compared to ANA- normals. Of these 33, 19 antigens were non-nuclear or cytoplasmic proteins and 14 were nuclear proteins, including known SLE associated antigens such as dsDNA and Histones. Antigen cluster analysis showed that DNA and RNA clusters are more frequent in SLE and ILE group, while matrix proteins and antiphospholipid antibodies clusters are more frequent in ANA+ normals. Next, all the individuals were genotyped with immunochip and subset by target sequencing. Quantitative genetic association test identified SNPs associated with ANA and auto-antigens. The strongest association signal was observed at HLA, where SNP rs3117103 near BTNL2, rs9268832 near HLA-DRA and rs2395252 in HLA-DQA2 gene showed strongest (10-7) association. Other signals were in/near BANK1, PTTG1 and CR1L genes. RNA sequencing analysis identified ANA risk alleles associated with up regulation of HLA-DRB1 in monocytes and BANK1 in B cells. Our data suggest that functional variations in or near HLA DRB1 and BANK1 gene may be associated with development of humoral autoimmunity in general population.

Discovering Relationships Between Lysine Ubiquitylation and Acetylation Sites from Proteomic Datasets

Ubiquitylation (Ub) and acetylation (Ac) are post translational modifications that both occur on lysine residues. Ac of non‐nuclear proteins is a new area of research, especially in the plant biology field. Ac of proteins can alter enzyme activities and protein functions, while Ub is well known for marking proteins for degradation by the proteasome as well as many other cellular functions. Recently, it has been suggested that competition may exist between Ub and Ac of specific lysine residues. It may be that the neutralization of the lysine side chain caused by Ac prevents Ub from taking place. If proven, this Ac‐Ub competition has the potential to affect processes such as the regulation of the cell cycle and protein degradation. The purpose of this study was to manually data mine previously published Ac and Ub sites in Arabidopsis thaliana to determine the amount of overlap. Sites identified in three publications were compared: Finkemeier et al (2011) Plant Physiol, 155:1779‐90; Kim et al (2013) The Plant Cell, 25: 1523‐40; and Wu et al (2011) Plant Physiol, 155:1769‐78. Thus far, seven of the modified proteins are subjected to both of these post‐translational modifications. Further analysis has been initiated to locate the specific Ub lysine residues and compare sites to known Ac lysine sites of the same protein. Currently, more extensive mass spectrometry based proteomics studies are underway to expand the number of identified Ac sites. Future work will investigate other plant species and other types of acylations. This project is part of a collaboration between The College of St. Scholastica and The University of Minnesota and provides research opportunities for undergraduates.

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

Abstract 3211: Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection.

Abstract Currently, proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue (the most common method of storing tissue specimens) are limited by the overwhelming complexity of the cellular proteome, which makes disease related changes in protein expression difficult to detect. Expression Microdissection (xMD) allows for automated, operator-independent dissection of specific cells or subcellular organelles in FFPE tissue, resulting in the enrichment of specific, selectable protein populations. In xMD, tissue slices that have been stained for specific cellular markers using immunohistochemical (HC) methods are placed under a layer of ethylene vinyl acetate (EVA) polymer film. When the stack is uniformly illuminated (using a rastering laser or a light pulse), the EVA melts and makes contact only over the stained tissue, enabling specific capture and transfer of these areas for further biochemical analysis, while leaving unstained tissue behind. As a proof of principle, we are using the xMD technique to enrich for nuclei in FFPE rat brain tissue sections followed by proteomic analysis. Shotgun proteomic analysis of these samples show a significant enrichment in nuclear localized proteins, with an average 18% of recovered proteins localized to the nucleus-versus 11% of recovered proteins in whole tissue scrapes. Targeted mass spectrometry, using Multiple Reaction Monitoring (MRM), also shows an enrichment of nuclear markers such as histones and DNA-binding proteins, and a de-enrichment of non-nuclear proteins. The enrichment for nuclei allows for the identification of methylated lysines on histones that are not detected in whole tissue scrapes, most likely due to their low abundance. The evolving methodology may allow researchers to profile the sub-cellular proteome of patient samples to identify potential new disease biomarkers and drug targets. This novel technology could also be used to advance our understanding of the biomolecular changes responsible for a specific disease state. Citation Format: Adele R. Blackler, Michael A. Tangrea, Tom Pohida, Nicole Morgan, Robert Bonner, Keshia Mora, John Kakareka, Sanford Markey, Michael R. Emmert-Buck. Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2013-3211

Prediction of nuclear proteins using nuclear translocation signals proposed by probabilistic latent semantic indexing

BackgroundIdentification of subcellular localization in proteins is crucial to elucidate cellular processes and molecular functions in a cell. However, given a tremendous amount of sequence data generated in the post-genomic era, determining protein localization based on biological experiments can be expensive and time-consuming. Therefore, developing prediction systems to analyze uncharacterised proteins efficiently has played an important role in high-throughput protein analyses. In a eukaryotic cell, many essential biological processes take place in the nucleus. Nuclear proteins shuttle between nucleus and cytoplasm based on recognition of nuclear translocation signals, including nuclear localization signals (NLSs) and nuclear export signals (NESs). Currently, only a few approaches have been developed specifically to predict nuclear localization using sequence features, such as putative NLSs. However, it has been shown that prediction coverage based on the NLSs is very low. In addition, most existing approaches only attained prediction accuracy and Matthew's correlation coefficient (MCC) around 54%~70% and 0.250~0.380 on independent test set, respectively. Moreover, no predictor can generate sequence motifs to characterize features of potential NESs, in which biological properties are not well understood from existing experimental studies.ResultsIn this study, first we propose PSLNuc (Protein Subcellular Localization prediction for Nucleus) for predicting nuclear localization in proteins. First, for feature representation, a protein is represented by gapped-dipeptides and the feature values are weighted by homology information from a smoothed position-specific scoring matrix. After that, we incorporate probabilistic latent semantic indexing (PLSI) for feature reduction. Finally, the reduced features are used as input for a support vector machine (SVM) classifier. In addition to PSLNuc, we further identify gapped-dipeptide signatures for putative NLSs and NESs to develop a prediction method, PSLNTS (Protein Subcellular Localization prediction using Nuclear Translocation Signals). We apply PLSI to generate gapped-dipeptide signatures from both nuclear and non-nuclear proteins, and propose candidate sequence motifs for putative NLSs and NESs. Then, we incorporate only the proposed gapped-dipeptide signatures in an SVM classifier to mimic biological properties of NLSs and NESs for predicting nuclear localization in PSLNTS.ConclusionsExperiment results demonstrate that the proposed method shows a significant improvement for nuclear localization prediction. To compare our predictive performance with other approaches, we incorporate two non-redundant benchmark data sets, a training set and an independent test set. Evaluated by five-fold cross-validation on the training set, PSLNuc attains an overall accuracy of 79.7%, which is 4.8% improvement over the state-of-the-art system. In addition, our method also enhances the MCC from 0.497 to 0.595. Compared on the independent test set, PSLNuc outperforms other predictors by 3.9%~19.9% on accuracy and 0.077~0.207 on MCC. This suggests that, in addition to NLSs, which have been shown important for nuclear proteins, NESs can also be an effective indicator to detect non-nuclear proteins. Most notably, using only a few proposed gapped-dipeptide signatures as input features for the SVM classifier, PSLNTS further enhances the accuracy and MCC to 80.9% and 0.618, respectively. Our results demonstrate that gapped-dipeptide signatures can better discriminate nuclear and non-nuclear proteins. Moreover, the proposed gapped-dipeptide signatures can be biologically interpreted and used in further experiment analyses of nuclear translocation signals, including NLSs and NESs.

Sumoylation of SAE2 C Terminus Regulates SAE Nuclear Localization

SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

Control of Seed Germination by Light-Induced Histone Arginine Demethylation Activity

For optimal survival, various environmental and endogenous factors should be monitored to determine the appropriate timing for seed germination. Light is a major environmental factor affecting seed germination, which is perceived by phytochromes. The light-dependent activation of phytochrome B (PHYB) modulates abscisic acid and gibberellic acid signaling and metabolism. Thus far, several negative regulators of seed germination that act when PHYB is inactive have been reported. However, neither positive regulators of seed germination downstream of PHYB nor a direct mechanism for regulation of the hormone levels has been elucidated. Here, we show that the histone arginine demethylases, JMJ20 and JMJ22, act redundantly as positive regulators of seed germination. When PHYB is inactive, JMJ20/JMJ22 are directly repressed by the zinc-finger protein SOMNUS. However, upon PHYB activation, JMJ20/JMJ22 are derepressed, resulting in increased gibberellic acid levels through the removal of repressive histone arginine methylations at GA3ox1/GA3ox2, which in turn promotes seed germination.

The emerging role of lysine acetylation of non-nuclear proteins.

Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors in the nucleus. More recent reports have also demonstrated that numerous proteins located outside the nucleus are also acetylated and that this modification has profound consequences on their functions. This review describes the latest findings on the substrates acetylated outside the nucleus and on the acetylases and deacetylates that catalyse these modifications. Protein acetylation is emerging as a major mechanism by which key proteins are regulated in many physiological processes such as migration, metabolism and aging as well as in pathological circumstances such as cancer and neurodegenerative disorders.

Characterisation of the passive permeability barrier of nuclear pore complexes

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve-like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross-linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant-negative importin β45-462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC-passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein-sized objects >10-fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.

Acetylation: a novel method for modulation of the immune response following trauma/hemorrhage and inflammatory second hit in animals and humans

Hemorrhage induces an imbalance in histone acetyl transferase/histone deacetylase (HAT/HDAC) ratio. Correction of this imbalance with histone deacetylase inhibitors (HDACI) improves survival. We aimed to identify whether this was due to modulation of the post-shock immune response. We established a "two-hit" model in which rats (n=11; 5-6/group) and humans (n=10; 5/group) sustained trauma/hemorrhage, followed by exposure of splenic leukocytes to lipopolysaccharide (LPS, 10 ng/mL) for 8 or 24 hours. The leukocytes were treated with: No treatment, SAHA (suberoylanilide hydroxamic acid, HDACI, 400 nM), or Garcinol (HAT inhibitor, 20 microM). Hemorrhage in the animals produced severe shock and a pro-inflammatory state. SAHA reduced TNFa secretion in the hemorrhaged leukocytes after LPS "second-hit" (34.0%, P = .003), whereas it increased transcript levels of TNFa and IL-1b (2.1+/-0.3 and 5.1+/- 2.2 fold respectively, P < .05). Leukocytes from trauma patients displayed 2 distinct responses to SAHA after LPS "second-hit," with markedly increased or decreased cytokine levels. SAHA normalizes TNFa levels following hemorrhage and LPS "second hit" in the rats, whereas trauma patients respond to SAHA in 2 distinct patterns, with either marked attenuation or exaggeration of inflammatory cytokines. Cytokine levels were independent of gene expression, implicating acetylation of non-nuclear proteins as the dominant regulatory mechanism.

Bcl-2 Localized at the Nuclear Compartment Induces Apoptosis after Transient Overexpression

Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.

Substrate and Functional Diversity of Lysine Acetylation Revealed by a Proteomics Survey

Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.