Rodrigo Gallardo

Increased Monomerization of Mutant HSPB1 Leads to Protein Hyperactivity in Charcot-Marie-Tooth Neuropathy

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.

Accurate Prediction of DnaK-Peptide Binding via Homology Modelling and Experimental Data

Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.

Structural diversity of PDZ-lipid interactions.

PDZ domains are globular protein modules that are over‐and‐above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ–phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin‐1, syntenin‐2, PTP‐Bas, PAR‐3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ–lipid interactions and the consequences for peptide interactions.

WALTZ-DB: a benchmark database of amyloidogenic hexapeptides

Accurate prediction of amyloid-forming amino acid sequences remains an important challenge. We here present an online database that provides open access to the largest set of experimentally characterized amyloid forming hexapeptides. To this end, we expanded our previous set of 280 hexapeptides used to develop the Waltz algorithm with 89 peptides from literature review and by systematic experimental characterisation of the aggregation of 720 hexapeptides by transmission electron microscopy, dye binding and Fourier transform infrared spectroscopy. This brings the total number of experimentally characterized hexapeptides in the WALTZ-DB database to 1089, of which 244 are annotated as positive for amyloid formation.

Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody

ABSTRACT The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. IMPORTANCE M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2.

A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover

Mutations in LRRK2 are a common cause of genetic Parkinson’s disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.The Parkinson’s disease‐associated LRRK2 protein is a multidomain Roco protein with GTPase activity. Here the authors use a multidisciplinary approach to characterize the GTPase mechanism of a homologous bacterial Roco protein and give mechanistic insights into disease-causing LRRK2 mutations.

De novo design of a biologically active amyloid

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science, this issue p. 10.1126/science.aah4949 A designed peptide drives a protein that does not usually aggregate to form amyloids. INTRODUCTION It has been shown that most proteins possess amyloidogenic segments. However, only about 30 human proteins are known to be involved in amyloid-associated pathologies, and it is still not clear what determines amyloid toxicity in these diseases. We investigated whether an endogenously expressed protein that contains sequences with known amyloidogenic segments, but is not known to aggregate either under normal or pathological conditions, can be induced to do so by seeding it with a peptide comprising the protein’s own amyloidogenic fragment. We chose to target the protein vascular endothelial growth factor receptor 2 (VEGFR2) because it has well-characterized biological function and so could provide a model system with which to investigate the relationship between protein loss of function and amyloid toxicity in different cellular contexts. RATIONALE The capacity of the amyloid conformation of disease proteins to catalyze their own amyloid conversion demonstrates the sequence specificity of amyloid assembly. Because the core of amyloids consists of short amyloidogenic sequence fragments, we hypothesized that a short amyloidogenic protein sequence of VEGFR2, a protein normally not associated with protein aggregation, should be able to interact with and specifically induce the aggregation of VEGFR2, resulting in its functional knockdown. We used TANGO, an algorithm that predicts aggregation-prone sequences, to identify potential amyloidogenic fragments in VEGFR2. We synthesized these fragments as a tandem repeat in a peptide framework in which each unit is flanked by charged residues and coupled by a short peptide linker. The thinking behind this design was that the tandem repeats would promote the formation of diffusable soluble oligomeric aggregates, whereas the charged residues would kinetically stabilize these oligomers and reduce the rate of insoluble fibril formation. RESULTS By screening for loss of function, we identified one peptide, termed “vascin,” that was highly potent at inhibiting VEGFR2. This sequence was derived from the translocation signaling sequence of VEGFR2. We found that vascin is an amyloidogenic peptide that readily forms small β-structured oligomers, ranging from dimers to nonamers, that slowly convert to amyloid fibrils. When added to cell culture medium, these oligomers are efficiently absorbed by the cell, where they interact with and promote the aggregation and partial degradation of nascent VEGFR2. Vascin aggregation does not induce the aggregation of known disease amyloids. Neither do vascin oligomers affect the function of the related EGF receptor or the surface translocation of other receptors. We found vascin only to be toxic to cells that are dependent on VEGFR2 function, suggesting that toxicity is due to loss of VEGFR2 function and not to vascin aggregation or vascin-induced VEGFR2 aggregation. Consistent with this, we found that vascin is active in vivo and could reduce tumor growth in a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor model in mice but is not intrinsically toxic to other tissues. CONCLUSION We found that a short amyloidogenic protein fragment can induce the aggregation of a protein normally not associated with amyloidosis in a manner that recapitulates key biophysical and biochemical characteristics of natural amyloids. In addition, we found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, rather than being generic, amyloid toxicity here appears to be both protein-specific and conditional on a requirement for VEGFR2 protein function. A synthetic amyloid peptide induces aggregation. We designed vascin, a synthetic amyloid peptide based on an amyloidogenic fragment of the signal peptide of VEGFR2. Vascin forms prefibrillar oligomers that penetrate mammalian cells and interacts with the nascent VEGFR2 protein, resulting in its aggregation and functional knockdown. [Composition includes parts of an image from iStock.com/luismmolina.] Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional—determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

Prediction and Reduction of the Aggregation of Monoclonal Antibodies

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.

Structural Hot Spots for the Solubility of Globular Proteins

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.

Sequence-dependent Internalization of Aggregating Peptides

Background: Prionoid propagation requires cell internalization of aggregated polypeptides. Results: Aggregates of different sequence are internalized through different endocytic pathways. Only phagocytosed aggregates (>1 μm) elicit an HSF1-dependent proteostatic response. Conclusion: Proteostatic response upon aggregate internalization differs markedly depending on the sequence. Significance: The characterization of mechanisms of cell penetration is fundamental for the understanding of aggregate transmission in disease. Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 μm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.