Federico Agostini

DnaK Functions as a Central Hub in the E. coli Chaperone Network

Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.

Predicting protein associations with long noncoding RNAs

Supplementary Figure 4 Predictions of interactions between PRC2 protein components and HOTAIR regions Supplementary Table 1 PDB IDs of non-redundant protein-RNA complexes used to train catRAPID Supplementary Table 2 Coefficients associated with protein and RNA properties Supplementary Table 3 Parameters of the interaction matrix I Supplementary Table 4 Composition of the NPInter dataset Supplementary Table 5 Composition of the Protein-binding (Protein BP), DNAbinding, RNA-binding (RNA BP) datasets Supplementary Table 6 Human MRP complex: Comparison between catRAPID predictions and experimental data Supplementary Methods

catRAPID omics: a web server for large-scale prediction of protein-RNA interactions

Summary: Here we introduce catRAPID omics, a server for large-scale calculations of protein–RNA interactions. Our web server allows (i) predictions at proteomic and transcriptomic level; (ii) use of protein and RNA sequences without size restriction; (iii) analysis of nucleic acid binding regions in proteins; and (iv) detection of RNA motifs involved in protein recognition. Results: We developed a web server to allow fast calculation of ribonucleoprotein associations in Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Xenopus tropicalis (custom libraries can be also generated). The catRAPID omics was benchmarked on the recently published RNA interactomes of Serine/arginine-rich splicing factor 1 (SRSF1), Histone-lysine N-methyltransferase EZH2 (EZH2), TAR DNA-binding protein 43 (TDP43) and RNA-binding protein FUS (FUS) as well as on the protein interactomes of U1/U2 small nucleolar RNAs, X inactive specific transcript (Xist) repeat A region (RepA) and Crumbs homolog 3 (CRB3) 3′-untranslated region RNAs. Our predictions are highly significant (P < 0.05) and will help the experimentalist to identify candidates for further validation. Availability: catRAPID omics can be freely accessed on the Web at http://s.tartaglialab.com/catrapid/omics. Documentation, tutorial and FAQs are available at http://s.tartaglialab.com/page/catrapid_group. Contact: gian.tartaglia@crg.eu

Sequence-Based Prediction of Protein Solubility

In order to investigate the relationship between the thermodynamics and kinetics of protein aggregation, we compared the solubility of proteins with their aggregation rates. We found a significant correlation between these two quantities by considering a database of protein solubility values measured using an in vitro reconstituted translation system containing about 70% of Escherichia coli proteins. The existence of such correlation suggests that the thermodynamic stability of the native states of proteins relative to the aggregate states is closely linked with the kinetic barriers that separate them. In order to create the possibility of conducting computational studies at the proteome level to investigate further this concept, we developed a method of predicting the solubility of proteins based on their physicochemical properties.

Disulfide Bonds Reduce the Toxicity of the Amyloid Fibrils Formed by an Extracellular Protein

The misfolding of proteins into amyloid fibrils constitutes the hallmark of many diseases.[1] Although relatively few physicochemical properties of protein sequences—charge, hydrophobicity, patterns of polar and nonpolar residues, and tendency to form secondary structures—are sufficient to rationalize in general terms their relative propensities to form amyloid fibrils,[2, 3] other properties can also be important. One example is intramolecular disulfide bonds, which limit the ways in which a polypeptide can be arranged in a fibril through the topological restraints that they impose. Although disulfide bonds are present in 15 % of the human proteome, in 65 % of secreted proteins, and in more than 50 % of those involved in amyloidosis, our understanding of how they influence the properties of amyloid fibrils is limited.[4–6] We have examined the formation of fibrils by human lysozyme[7, 8] in the presence and absence (Figure 1 a,b) of its native disulfide bonds, and found that they profoundly influence the fibrillar morphology and cytotoxicity. Figure 1 a) Structure (pdb code 1Lz1) of wild-type lysozyme (Lys) with the disulfide bonds shown in red. These were reduced as shown in (b) to obtain LysRA. c, d) Amyloid formation by Lys (c) and LysRA (d) monitored by light scattering (LS) at 500 nm and different ... As disulfide bonds stabilize folded proteins, they determine the conditions under which wild-type (Lys) and reduced and alkylated lysozyme (LysRA) are amyloidogenic. In agreement with previous reports, we found that it is necessary to incubate Lys under destabilizing conditions, such as low pH (pH 2.0) and high temperature (≥50 °C), to form amyloid fibrils within 24 h (Figure 1 c and Figure S1 in the Supporting Information).[8–10] By contrast, LysRA is amyloidogenic under milder conditions; at pH 2.0, for example, it forms fibrils at 20 °C (Figure 1 d and Figure S1 in the Supporting Information). We analyzed the conformational properties of Lys and LysRA by NMR spectroscopy and far-UV circular dichroism (CD) as a function of temperature. We found that Lys is folded at 20 °C (Figure 1 e,g) and experiences a well-defined unfolding transition at about 55 °C (Figure 1 g and Figure S2 in the Supporting Information).[10] By contrast, LysRA is unfolded at all temperatures (Figure 1 f,h). Our results, therefore, indicate that the presence of intact disulfide bonds decreases the rate at which lysozyme forms fibrils (Figure 1 c,d) by stabilizing the cooperatively folded native protein.[11] Disulfide bonds also determine the morphology of the fibrils. After 24 h of incubation under the mildest conditions that lead to aggregation (Figure 1 c,d), both Lys and LysRA had converted into fibrils as shown by transmission electron microscopy (TEM; Figure 2 a,b insets) and by thioflavin T (ThT) and Congo red (CR) binding (Figure 2 c,d and e,f, respectively). We analyzed the samples by far-UV CD and found that in both cases the spectra evolved from those corresponding to largely disordered proteins (Figure 2 a,b, blue) to those of species rich in β-sheet structure, with a minimum in the ellipticity at approximately 217 nm typical of amyloid fibrils (Figure 2 a,b, red). We also analyzed the amide I region (1580–1720 cm−1) of the infrared spectra of the fibrils by using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy (Figure 3 a,b and Table S1 in the Supporting Information),[9] and found that LysRA fibrils are less rich in β-sheet structure than those formed by Lys (51.5 versus 72.5 %). Figure 2 Aggregation kinetics of a) Lys at pH 2.0 and 60 °C and b) LysRA at pH 2 and 20 °C monitored by far UV-CD. Samples of fibrils formed after 24 h and isolated by ultracentrifugation have a fibrillar morphology (insets in (a) and (b)) and ... Figure 3 a, b) ATR-FTIR spectra of LysRA (a) and Lys fibrils (b), shown in black, with the contributions obtained by curve fitting colored as follows: red: β sheet, green: random/α helix, blue: turns and loops, gray: side chains. c) SDS-PAGE of ... To analyze the nature of the fibrillar core of the fibrils, we studied both types of fibrils by limited proteolysis (Figure 3 c,d). It was found that the fibrils formed by Lys are inert to proteolysis under the conditions used here but that those formed by LysRA are readily cleaved (Figure 3 c,d and Figure S3 in the Supporting Information) and have their diameter reduced from (5.1±0.6) to (3.6±0.6) nm (Figure 2 b and Figure S4 in the Supporting Information). The protease-resistant segment of the molecule, attributable to the core, is composed of about 80 residues, from residue 29 to 108. As susceptibility to proteases requires 10 to 12 unfolded residues,[12] our result is consistent with the number of residues in the β-sheet secondary structure determined by FTIR analysis (51.5 %, that is, 67 residues). We also probed the nature of the non-core regions by an 8-anilinonaphthalene-1-sulfonate (ANS) binding assay, in which interactions of this dye with solvent-exposed hydrophobic patches cause a blue shift in the maximum emission wavelength and an increase in emission intensity.[13] We found that the fluorescence intensity of ANS is higher in the presence of LysRA fibrils than in that of fibrils formed from Lys (Figure S5 in the Supporting Information), hence indicating a greater number of solvent-exposed hydrophobic residues. Since hydrogen-bonding interactions in the cross-β core stabilize amyloid fibrils,[14] we investigated whether differences in core size are reflected in their resistance to disaggregation. We measured the concentration of protein in equilibrium with fibrils at increasing concentrations of guanidine hydrochloride (GdnHCl)[15] and found that the fibrils formed by LysRA disaggregate at lower concentrations of GdnHCl than those formed by Lys (Figure 3 e). Our results indicate that the fibrillar core formed in the presence of disulfide bonds is larger than in their absence, thereby reducing the susceptibility of the fibrils to proteolysis and increasing their stability. Current evidence suggests that the most toxic forms of amyloid aggregates are not the mature fibrils but their less organized precursors.[16] In addition, recent studies have shown that partially structured fibrils can also give rise to toxicity as a result of their larger accessible hydrophobic area or by their greater tendency to generate toxic oligomeric species by fragmentation.[9] To investigate whether or not disulfide bonds alter the cytotoxicity of the fibrils, samples corresponding to protein concentrations of 5 to 20 μm were added to cultures of SH-SY5Y human neuroblastoma cells and the resulting changes in cell viability were measured using a calcein acetoxymethyl (AM) assay (Figure 3 f). The results, supported by an MTT assay (MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Figure S6 in the Supporting Information), show that the fibrils formed by LysRA have a significantly higher cytotoxic effect than those formed by Lys (p<0.0001; Figure 3 f), in agreement with the finding that ANS binding in amyloid species (Figure S5 in the Supporting Information) correlates with cytotoxicity.[17] This result shows that disulfide bonds can decrease toxicity by favoring the formation of highly structured amyloid fibrils, which suggests that disulfide bonds in extracellular proteins could be the result of evolutionary pressures[18] to minimize toxic aggregation in an environment where the redox potential favors disulfide bond formation. To investigate this hypothesis further, we analyzed the aggregation propensity of the human proteome using the well-established Zyggregator predictor.[3] We found that the sequences of extracellular proteins have higher intrinsic aggregation propensity than intracellular ones, an observation that has been related to the dilution that occurs upon secretion.[19, 20] We also found that disulfide bonds are associated with sequences of high aggregation propensity (Figure 3 g), which suggests that disulfide bonds have co-evolved with protein sequences[21] to minimize their propensity to form potentially toxic amyloid aggregates. This analysis would explain the high prevalence of disulfide bonds in extracellular proteins, where additional protective mechanisms that reduce misfolding and its consequences are likely to play a less significant role than inside the cell.[20, 22] The disulfide bonds of lysozyme inhibit the aggregation of this protein into amyloid fibrils by stabilizing the folded state—a fact that can be attributed to the reduction in the entropy of the unfolded state.[11] A partially unfolded state can nevertheless be populated as a result of changes in conditions, as in this work, or by mutations, as in patients with nonneuropathic systemic amyloidosis.[8] We have shown that, when this situation occurs, disulfide bonds allow the formation of fibrils with a large proportion of their sequence in the cross-β conformation. This result is at first sight unexpected, as one might have anticipated that the conformational constraints resulting from cross-linking would reduce the ability of the chain to fold into the complex β-sheet amyloid structure. It is, however, clear that lysozyme and other disulfide-linked proteins are able to form fibrils that contain a high fraction of sequence in the cross-β structure.[23] We conclude that intramolecular disulfide bonds can stabilize amyloid fibrils, as they do for the folded state, by decreasing the entropic penalty associated with the formation of this ordered form of protein structure. This can be concomitant with significant decreases in the toxicity of the resulting fibrils, which suggests that disulfide bonds have co-evolved with protein sequences to reduce toxic aggregation.[21]

Neurodegenerative diseases: Quantitative predictions of protein–RNA interactions

Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimers, and Parkinsons diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

Principles of self-organization in biological pathways: a hypothesis on the autogenous association of alpha-synuclein

Previous evidence indicates that a number of proteins are able to interact with cognate mRNAs. These autogenous associations represent important regulatory mechanisms that control gene expression at the translational level. Using the catRAPID approach to predict the propensity of proteins to bind to RNA, we investigated the occurrence of autogenous associations in the human proteome. Our algorithm correctly identified binding sites in well-known cases such as thymidylate synthase, tumor suppressor P53, synaptotagmin-1, serine/ariginine-rich splicing factor 2, heat shock 70 kDa, ribonucleic particle-specific U1A and ribosomal protein S13. In addition, we found that several other proteins are able to bind to their own mRNAs. A large-scale analysis of biological pathways revealed that aggregation-prone and structurally disordered proteins have the highest propensity to interact with cognate RNAs. These findings are substantiated by experimental evidence on amyloidogenic proteins such as TAR DNA-binding protein 43 and fragile X mental retardation protein. Among the amyloidogenic proteins, we predicted that Parkinson’s disease-related α-synuclein is highly prone to interact with cognate transcripts, which suggests the existence of RNA-dependent factors in its function and dysfunction. Indeed, as aggregation is intrinsically concentration dependent, it is possible that autogenous interactions play a crucial role in controlling protein homeostasis.

Constitutive patterns of gene expression regulated by RNA-binding proteins

BackgroundRNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized.ResultsWe combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server.ConclusionsOur analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies.

X-inactivation: quantitative predictions of protein interactions in the Xist network

The transcriptional silencing of one of the female X-chromosomes is a finely regulated process that requires accumulation in cis of the long non-coding RNA X-inactive-specific transcript (Xist) followed by a series of epigenetic modifications. Little is known about the molecular machinery regulating initiation and maintenance of chromosomal silencing. Here, we introduce a new version of our algorithm catRAPID to investigate Xist associations with a number of proteins involved in epigenetic regulation, nuclear scaffolding, transcription and splicing processes. Our method correctly identifies binding regions and affinities of protein interactions, providing a powerful theoretical framework for the study of X-chromosome inactivation and other events mediated by ribonucleoprotein associations.

Predictions of protein–RNA interactions

Ribonucleoprotein interactions play important roles in a wide variety of cellular processes, ranging from transcriptional and posttranscriptional regulation of gene expression to host defense against pathogens. High throughput experiments to identify RNA–protein interactions provide information about the complexity of interaction networks, but require time and considerable efforts. Thus, there is need for reliable computational methods for predicting ribonucleoprotein interactions. In this review, we discuss a number of approaches that have been developed to predict the ability of proteins and RNA molecules to associate.