Pathway-based biomarker search by high-throughput proteomics profiling of secretomes.

An efficient means for the identification of prognostic and predictive biomarkers is essential in today's cancer management. A new approach toward biomarker discovery has therefore been proposed, where pathways instead of individual proteins would be monitored and targeted. Recently, the 'secretome', a biological fluid that may be enriched with secreted and/or shed proteins from adjacent disease-relevant cancer cells, has been targeted for biomarker discovery. We describe a novel method for secretome analysis using "stacking gels", label-free relative quantitation, and pathway analysis. The protocol presented here increases the throughput of secretome analysis by approximately 1 order of magnitude compared to earlier methodologies. In the first application, six cancer cell lines from three different tissues were studied. The global secretome data sets obtained were analyzed using pathway analysis software to attempt integrating the experimental findings into a cellular signaling context. This suggested that several secretome proteins might be interconnected with intracellular canonical pathways. This, in turn, may eventually allow the use of secretomes for discovery of pathway-based biomarkers. When this strategy was applied to two breast cancer cell lines, it appeared that the IGF signaling and the plasminogen activating system may be differentially regulated in invasive breast cancer, but this remains speculative until it is verified in a clinical setting. In summary, the methodology proposed optimizes cell culture with sample fractionation and LC-MS to obtain the highest yield from cultured cell secretomes, with a focus on rational biomarker discovery through putative linkage with cancer relevant pathways.

PU.1 and a TTTAAA element in the myeloid defensin-1 promoter create an operational TATA box that can impose cell specificity onto TFIID function.

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein ("D1BP"), acting through, respectively, proximal (-22/-19) and distal (-62/-59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)alpha/GABPbeta. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking "TTTAAA" element (-32/-27), a "weak" but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a "strong" TATA box ("TATAAA") eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.

A direct interaction between the RAG2 C terminus and the core histones is required for efficient V(D)J recombination.

V(D)J recombination is a tightly controlled process of somatic recombination whose regulation is mediated in part by chromatin structure. Here, we report that RAG2 binds directly to the core histone proteins. The interaction with histones is observed in developing lymphocytes and within the RAG1/RAG2 recombinase complex in a manner that is dependent on the RAG2 C terminus. Amino acids within the plant homeo domain (PHD)-like domain as well as a conserved acidic stretch of the RAG2 C terminus that is considered to be a linker region are important for this interaction. Point mutations that disrupt the RAG2-histone association inhibit the efficiency of the V(D)J recombination reaction at the endogenous immunoglobulin locus, with the most dramatic effect in the V to DJ(H) rearrangement.

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.

Cytosol-derived proteins are sufficient for Arp2/3 recruitment and ARF/coatomer-dependent actin polymerization on Golgi membranes.

The actin cytoskeleton has been implicated in protein trafficking at the Golgi apparatus and in Golgi orientation and morphology. Actin dynamics at the Golgi are regulated in part by recruiting Cdc42 or Rac to the membrane through a binding interaction with the coatomer-coated (COPI)-vesicle coat protein, coatomer. This leads to actin polymerization through the effector, N-WASP and the Arp2/3 complex. Here, we have used reconstitution of vesicle budding to test whether Arp2/3 is recruited to membranes during the formation of COPI vesicles. Our results revealed that ARF1 activation leads to greatly increased Arp3 levels on the membranes. Coatomer-bound Cdc42 and pre-existing F-actin are important for Arp2/3 binding. ARF1-dependent Arp2/3 recruitment and actin polymerization can be reconstituted on liposomal membranes, indicating that no membrane proteins are necessary. These results show that activated ARF1 can stimulate Arp2/3 recruitment to Golgi membranes through coatomer, Cdc42 or Rac, and N-WASP.

Mutual targeting of mediator and the TFIIH kinase Kin28.

In Saccharomyces cerevisiae, Kin28 is a member of the cyclin-dependent kinase family. Kin28 is a subunit of the basal transcription factor holo-TFIIH and its trimeric sub-complex TFIIK. Kin28 is the primary kinase that phosphorylates the RNA polymerase II (RNA pol II) C-terminal domain (CTD) within a transcription initiation complex. Mediator, a global transcriptional co-activator, dramatically enhances the phosphorylation of the CTD of RNA pol II by holo-TFIIH in vitro. Using purified proteins we have determined that the subunits of TFIIK are sufficient for Mediator to enhance Kin28 CTD kinase activity and that Mediator enhances phosphorylation of a glutathione S-transferase-CTD fusion protein, despite the absence of multiple Mediator and/or TFIIH interactions with polymerase. Mediator does not stimulate the activity of several other CTD kinases, suggesting that the specific enhancement of TFIIH kinase activity results in Kin28 being the primary CTD kinase at initiation. In addition, we have found that Kin28 phosphorylates Mediator subunit Med4 in an assay, including purified holo-TFIIH, and either Mediator or recombinant Med4 alone. Furthermore, Kin28 appears to be, at least in part, responsible for the phosphorylation of Med4 in vivo. We have identified Thr-237 as the site of phosphorylation of Med4 by Kin28 in vitro. The mutation of Thr-237 to Ala has no effect on the growth of a yeast strain under normal conditions but confirms that Thr-237 is also the site of Med4 phosphorylation in vivo.

mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad.

The role of hSWI/SNF complexes in transcriptional activation is well characterized; however, little is known about their function in transcriptional repression. We have previously shown that subunits of the mSin3A/histone deacetylase 2 (HDAC2) corepressor complex copurify with hSWI/SNF complexes. Here we show that the type II arginine-specific methyltransferase PRMT5, which is involved in cyclin E repression, can be found in association with Brg1 and hBrm-based hSWI/SNF complexes. We also show that hSWI/SNF-associated PRMT5 can methylate hypoacetylated histones H3 and H4 more efficiently than hyperacetylated histones H3 and H4. Protein-protein interaction studies indicate that PRMT5 and mSin3A interact with the same hSWI/SNF subunits as those targeted by c-Myc. These observations prompted us to examine the expression profile of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (cad) and nucleolin (nuc). We found that cad repression is altered in cells that express inactive Brg1 and in cells treated with the HDAC inhibitor depsipeptide. Using chromatin immunoprecipitation assays, we found that Brg1, mSin3A, HDAC2, and PRMT5 are directly recruited to the cad promoter. These results suggest that hSWI/SNF complexes, through their ability to interact with activator and repressor proteins, control expression of genes involved in cell growth and proliferation.

The yeast capping enzyme represses RNA polymerase II transcription.

Using a highly pure transcription system derived from Saccharomyces cerevisiae, we have purified an activity in yeast whole-cell extracts that represses RNA polymerase II transcription. Mechanistic studies suggest that this repressor specifically targets transcriptional reinitiation. The two polypeptides that constitute the repressor have been identified as Ceg1p and Cet1p, the two subunits of the yeast pre-mRNA capping enzyme. A purified recombinant capping enzyme is able to reconstitute repressor activity. Cet1p is necessary for and capable of this repression. Transcriptional run-on experiments indicate that the capping enzyme also serves as a repressor in vivo. Efficient pre-mRNA capping relies on interactions between the capping enzyme and transcription apparatus. Repression by the capping enzyme suggests a bidirectional flow of information between capping and transcription.

Isolation and mass spectrometry of transcription factor complexes.

Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary approaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification of three novel subunits.

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.