The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module.

Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.

Comprehensive identification of disulfide bonds using non-specific proteinase K digestion and CID-cleavable crosslinking analysis methodology for Orbitrap LC/ESI-MS/MS data.

Disulfide bonds are valuable constraints in protein structure modeling. The Cys-Cys disulfide bond undergoes specific fragmentation under CID and, therefore, can be considered as a CID-cleavable crosslink. We have recently reported on the benefits of using non-specific digestion with proteinase K for inter-peptide crosslink determination. Here, we describe an updated application of our CID-cleavable crosslink analysis software and our crosslinking analysis with non-specific digestion methodology for the robust and comprehensive determination of disulfide bonds in proteins, using Orbitrap LC/ESI-MS/MS data.

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPß). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPß prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPß and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.

An isotopically coded CID-cleavable biotinylated cross-linker for structural proteomics.

Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.

Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks.

Cross-linking combined with mass spectrometry has great potential for determining three-dimensional structures of proteins and protein assemblies. One of the main analytical challenges of this method is the specific detection and identification of the inter-peptide crosslinks in the peptide mixture after enzymatic digestion of the cross-linked protein complex. These inter-peptide crosslinks are important because they provide the critical distance information needed for structural proteomics studies. In this paper, we demonstrate the use of isotopically coded N-terminal modification (ICNTM) in combination with isotopically coded cross-linkers (ICCL) for specific detection of inter-peptide crosslinks. Inter-peptide crosslinks contain two amino termini, compared to one in the case of free peptides, dead-end crosslinks, or intra-peptide crosslinks. Therefore, N-terminal modification with a 1:1 mixture of heavy and light isotopically coded reagents produces inter-peptide crosslinks with a distinct isotopic signature (a 1:2:1 ratio). Modification also occurs at the epsilon-amino groups of non-cross-linked lysine residues, resulting in two modifications per free lysine-containing peptide. However, if ICCL and ICNTM are used together, inter-peptide crosslinks can be distinguished from free lysine-containing peptides. Specialized software has also been developed for the analysis of ICCL + ICNTM experimental data. This procedure, combined with software for data analysis, provides a simple and rapid method for specific detection of inter-peptide crosslinks.