Anna Malovannaya

Analysis of the Human Endogenous Coregulator Complexome

Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset, we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687, and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), complex isoforms (uniCOREs), and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of future hypotheses.

The SRC-3/AIB1 Coactivator Is Degraded in a Ubiquitin- and ATP-Independent Manner by the REGγ Proteasome

Steroid receptor coactivator-3 (SRC-3/AIB1) is an oncogene frequently amplified and overexpressed in breast cancers. Here we report that SRC-3 interacts with REGgamma, a proteasome activator known to stimulate the trypsin-like activity of the 20S proteasome. RNAi knockdown and gain-of-function experiments suggest that REGgamma promotes SRC-3 protein degradation. Cellular levels of REGgamma expression affect estrogen-receptor target-gene expression and cell growth as a result of its ability to promote degradation of the SRC-3 protein. In vitro proteasome proteolysis assays using purified REGgamma, SRC-3, and the 20S proteasome reinforce these conclusions and demonstrate that REGgamma promotes the degradation of SRC-3 in a ubiquitin- and ATP-independent manner. This work demonstrates the first example of a physiologically relevant endogenous cellular target for the REGgamma-proteasome complex. It also highlights the fact that an alternative mode of proteasome-mediated protein degradation, independent of the 19S proteasome regulatory cap, targets the SRC-3 protein for degradation.

Streamlined analysis schema for high-throughput identification of endogenous protein complexes

Immunoprecipitation followed by mass spectrometry (IP/MS) has recently emerged as a preferred method in the analysis of protein complex components and cellular protein networks. Targeting endogenous protein complexes of higher eukaryotes, particularly in large-scale efforts, has been challenging due to cellular heterogeneity, high proteome complexity, and, compared to lower organisms, lack of efficient in-locus epitope-tagging techniques. It is further complicated by variability in nonspecific identifications and cross-reactivity of primary antibodies. Still, the study of endogenous human protein networks is highly desired despite its challenges. Here we describe a streamlined IP/MS protocol for the purification and identification of extended endogenous protein complexes. We investigate the sources of nonspecific protein binding and develop semiquantitative specificity filters that are based on peptide spectral count measurements. We also outline logical constraints for the derivation of accurate complex composition from IP/MS data and demonstrate the effectiveness of this approach by presenting our analyses of different transcriptional coregulator complexes. We show consistent purification of novel components for the Integrator complex, analyze the composition of the Mediator complex solely from our data to demonstrate the wide usability of spectral counts, and deconvolute heterogeneous HDAC1/2 networks into core complex modules and several novel subcomplex interactions.

A Data Set of Human Endogenous Protein Ubiquitination Sites

Lysine ubiquitination is an important and versatile protein post-translational modification. Numerous cellular functions are regulated by ubiquitination, suggesting that extensive numbers of proteins, if not all, are modified with ubiquitin at certain times. However, proteome-wide profiling of ubiquitination sites in the mammalian system is technically challenging. We report the design and characterization of an engineered protein affinity reagent for the isolation of ubiquitinated proteins and the identification of ubiquitination sites with mass spectrometry. This recombinant protein consists of four tandem repeats of ubiquitin-associated domain from UBQLN1 fused to a GST tag. We used this GST-qUBA reagent to isolate polyubiquitinated proteins and identified 294 endogenous ubiquitination sites on 223 proteins from human 293T cells without proteasome inhibitors or overexpression of ubiquitin. Mitochondrial proteins constitute 14.7% of this data set, implicating ubiquitination in a wide range of mitochondrial functions.

A Fast Workflow for Identification and Quantification of Proteomes

The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.

OTU Domain-containing Ubiquitin Aldehyde-binding Protein 1 (OTUB1) Deubiquitinates Estrogen Receptor (ER) α and Affects ERα Transcriptional Activity

Estrogen receptor (ER) α is an essential component in human physiology and is a key factor involved in the development of breast and endometrial cancers. ERα protein levels and transcriptional activity are tightly controlled by the ubiquitin proteasome system. Deubiquitinating enzymes, a class of proteases capable of removing ubiquitin from proteins, are increasingly being seen as key modulators of the ubiquitin proteasome system, regulating protein stability and other functions by countering the actions of ubiquitin ligases. Using mass spectrometry analysis of an ERα protein complex, we identified OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) as a novel ERα-interacting protein capable of deubiquitinating ERα in cells and in vitro. We show that OTUB1 negatively regulates transcription mediated by ERα in transient reporter gene assays and transcription mediated by endogenous ERα in Ishikawa endometrial cancer cells. We also show that OTUB1 regulates the availability and functional activity of ERα in Ishikawa cells by affecting the transcription of the ERα gene and by stabilizing the ERα protein in the chromatin.

Global characterization of transcriptional impact of the SRC-3 coregulator.

The nuclear receptor and bona fide oncogene, steroid receptor coactivator-3 (SRC-3, AIB1), acts as a master transcriptional regulator of breast cancer by transducing growth signals via the estrogen receptor alpha (ER). In this resource paper, we present the genome-wide localization analysis of SRC-3 chromatin affinity sites in MCF-7 human breast cancer chromatin and compare the cis binding sites to global cartographies for ER and FoxA1. By correlating their gene proximal binding sites to integrated gene expression signatures, and in combination with gene ontology analyses, we provide a functional classification of estradiol-induced gene regulation that further highlights an intricate transcriptional control of interdependent cellular pathways by SRC-3. Furthermore, by presenting proteomics analyses of in vivo SRC-3- and ER-associated proteins, we give strong evidence to support the idea that the interpretative power of SRC-3 in estrogen signaling is mediated through the formation of distinct, cell state-dependent protein complexes. Altogether, we present the first approach in complementary comparative analyses that converges results obtained by three discovery-driven methods (cistromics, transcriptomics, and proteomics) into testable hypotheses, thus providing a valuable resource for follow-up studies that further our understanding of estrogen signaling in human diseases in general and breast cancer in particular.

Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements.

Transcription factors (TFs) are families of proteins that bind to specific DNA sequences, or TF response elements (TFREs), and function as regulators of many cellular processes. Because of the low abundance of TFs, direct quantitative measurement of TFs on a proteome scale remains a challenge. In this study, we report the development of an affinity reagent that permits identification of endogenous TFs at the proteome scale. The affinity reagent is composed of a synthetic DNA containing a concatenated tandem array of the consensus TFREs (catTFRE) for the majority of TF families. By using catTFRE to enrich TFs from cells, we were able to identify as many as 400 TFs from a single cell line and a total of 878 TFs from 11 cell types, covering more than 50% of the gene products that code for the DNA-binding TFs in the genome. We further demonstrated that catTFRE pull-downs could quantitatively measure proteome-wide changes in DNA binding activity of TFs in response to exogenous stimulation by using a label-free MS-based quantification approach. Applying catTFRE on the evaluation of drug effects, we described a panoramic view of TF activations and provided candidates for the elucidation of molecular mechanisms of drug actions. We anticipate that the catTFRE affinity strategy will find widespread applications in biomedical research.

Proteomic analyses reveal distinct chromatin- associated and soluble transcription factor complexes

The current knowledge on how transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated by protein–protein interactions remains limited. Here, we performed proteomics analyses of soluble and chromatin‐associated complexes of 56 TFs, including the targets of many signalling pathways involved in development and cancer, and 37 members of the Forkhead box (FOX) TF family. Using tandem affinity purification followed by mass spectrometry (TAP/MS), we performed 214 purifications and identified 2,156 high‐confident protein–protein interactions. We found that most TFs form very distinct protein complexes on and off chromatin. Using this data set, we categorized the transcription‐related or unrelated regulators for general or specific TFs. Our study offers a valuable resource of protein–protein interaction networks for a large number of TFs and underscores the general principle that TFs form distinct location‐specific protein complexes that are associated with the different regulation and diverse functions of these TFs.

Proteogenomic integration reveals therapeutic targets in breast cancer xenografts.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.