Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome

Abstract

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.

Figure 1. Percentage of proteins identified as DPs, MXPs and FPs in control and heat-treated samples from mouse fibroblasts

The percentages of proteins classified as DPs (blue bars), MXPs (open bars), and FPs (orange bars) in datasets derived from 2D PAGE analyses of untreated (left) and heat-treated (center) mouse fibroblast cell extracts are compared to those determined through MudPIT analysis (right) of a similar heat-treated extract. The total number of proteins detected in each experiment is shown at the top in parentheses.

Figure 2. Percentage of proteins classified as DPs, MXPs and FPs in the mouse TS protein dataset that contain known sites of post-translational modification (PTM) and phosphorylation, alternate splice variants or transmembrane domains

Key: blue bars, PTMs; red bars, phosphorylation; black bars, alternative splice variants; and white bars, transmembrane domains. The total number of proteins in each structural class is shown at the top in parentheses.

Figure 3

Biological functions associated with disordered (DPs + MXPs) and folded/ordered proteins (FPs) in TS dataset. Graphical representation of over- and under-representation of GO terms for disordered proteins (DPs + MXPs) and folded ordered proteins (FPs) for three functional categories, (A) cell component, (B) biological process, and (C) molecular function. Results are shown only for over- and under-represent GO terms with false discover rate (FDR) values <0.01 and with ≥10 associated proteins. The column labeled Background indicates the percentage of all theoretical mouse proteins that exhibited a particular GO term using a gray scale. The columns labeled (DPs + MXPs) and FPs indicate the extent of over- (red scale) or under-representation (green scale) of a particular GO term, given as [(LH/LT) – (BH/BT)]/(BH/BT) × 100; where: LH is the number of disordered or folded proteins associated with a particular GO term, LT is the total number of disordered or folded proteins with any GO term, BH is the number of theoretical mouse proteins associated with a particular GO term, and BT is the number of theoretical mouse proteins associated with any GO term. The color and gray scales are defined in the lower right. Gray boxes in the columns labeled (DPs + MXPs) and FPs indicate that the noted GO term was not over- or under-represented for the indicated structural class and have the same shade as the box labeled Background; asterisks in the columns labeled (DPs + MXPs) and FPs indicate that zero proteins in the indicated structural class were associated with the noted GO term.

Figure 4. Analysis of the number of protein interaction partners for proteins in the different structural classes in the TS protein dataset

The percentage of DPs (blue diamonds), MXPs (black squares) and FPs (orange circles) which interact with up to the given numbers of interaction partners is plotted versus the number of interaction partners. The boxed region is expanded in the upper right. The data represent totals over bins incremented by 5 interaction partners (e.g., 0-5 partners, 6-10 partners, etc.).

Figure 5. Cooperation amongst intrinsically disordered and coiled-coil segments within IC promotes binding to LC8

(A) A short interaction motif (IM) from two molecules of the intrinsically disordered protein, IC (residues 84-260 illustrated), adopt rigid, extended structure when bound on opposite faces of the folded, dimeric protein, LC8. While not directly involved in binding to LC8, two leucine-zipper (LZ) motif-containing segments of IC, that are unfolded and monomeric in the absence of LC8, form a coiled-coil dimer when the IM segments of IC bind to LC8. IC is illustrated as a yellow tube, with the IM segments and LZ motifs colored red or green, respectively, in the two molecules. The two subunits of the LC8 dimer are shown in surface representation in dark and light blue, respectively. (B) The LC8 dimer was rotated 90° relative to (A) and only the IM segments of the two IC molecules are illustrated as red and green tubes, respectively. [Modeled after Figure 2 in ref. 69, with permission from the author.]