Francesc X. Avilés

AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides

BackgroundProtein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimers and Parkinsons diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets.ResultsAGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies.ConclusionBy identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/ shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimers and Parkinsons diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets. AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies. By identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/ shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.

Metallocarboxypeptidases and their protein inhibitors: Structure, function and biomedical properties

Among the different aspects of recent progress in the field of metallocarboxypeptidases has been the elucidation of the three dimensional structures of the pro-segments (in monomeric or oligomeric species) and their role in the expression, folding and inhibition/activation of the pancreatic and pancreatic-like forms. Also of great significance has been the cloning and characterization of several new regulatory carboxypeptidases, enzymes that are related with important functions in protein and peptide processing and that show significant structural differences among them and also with the digestive ones. Many regulatory carboxypeptidases lack a pro-region, unlike the digestive forms or others in between from the evolutionary point of view. Finally, important advances have been made on the finding and characterization of new protein inhibitors of metallocarboxypeptidases, some of them with interesting potential applications in the biotechnological/biomedical fields. These advances are analyzed here and compared with the earlier observations in this field, which was first explored by Hans Neurath and collaborators.

Inclusion bodies : Specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

Mutagenesis of the central hydrophobic cluster in Aβ42 Alzheimer's peptide

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. Alzheimers disease is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain, composed primarily of the 42 amino acid human β‐amyloid peptide (Aβ42). Disease‐linked mutations in Aβ42 occur in or near a central hydrophobic cluster comprising residues 17–21. We exploited the ability of green fluorescent protein to act as a reporter of the aggregation of upstream fused Aβ42 variants to characterize the effects of a large set of single‐point mutations at the central position of this hydrophobic sequence as well as substitutions linked to early onset of the disease located in or close to this region. The aggregational properties of the different protein variants clearly correlated with changes in the intrinsic physicochemical properties of the side chains at the point of mutation. Reduction in hydrophobicity and beta‐sheet propensity resulted in an increase of in vivo fluorescence indicating disruption of aggregation, as confirmed by the in vitro analysis of synthetic Aβ42 variants. The results confirm the key role played by the central hydrophobic stretch on Aβ42 deposition and support the hypothesis that sequence tunes the aggregation propensities of polypeptides.

The three-dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen

The three‐dimensional structure of human procarboxypeptidase A2 has been determined using X‐ray crystallography at 1.8 Å resolution. This is the first detailed structural report of a human pancreatic carboxypeptidase and of its zymogen. Human procarboxypeptidase A2 is formed by a pro‐segment of 96 residues, which inhibits the enzyme, and a carboxypeptidase moiety of 305 residues. The pro‐enzyme maintains the general fold when compared with other non‐human counterparts. The globular part of the pro‐segment docks into the enzyme moiety and shields the S2‐S4 substrate binding sites, promoting inhibition. Interestingly, important differences are found in the pro‐segment which allow the identification of the structural determinants of the diverse activation behaviours of procarboxypeptidases A1, B and A2, particularly of the latter. The benzylsuccinic inhibitor is able to diffuse into the active site of procarboxypeptidase A2 in the crystals. The structure of the zymogen‐inhibitor complex has been solved at 2.2 Å resolution. The inhibitor enters the active site through a channel formed at the interface between the pro‐segment and the enzyme regions and interacts with important elements of the active site. The derived structural features explain the intrinsic activity of A1/A2 pro‐enzymes for small substrates.

A novel subfamily of mouse cytosolic carboxypeptidases

Nnal is a recently described gene product that has sequence similarity with metallocar‐boxypeptidases. In the present study, five additional Nnal‐like genes were identified in the mouse genome and named cytosolic carboxypeptidase (CCP) 2 through 6. Modeling suggests that the carboxypeptidase domain folds into a structure that resembles metallocarboxypeptidases of the M14 family, with all necessary residues for catalytic activity and broad substrate specificity. All CCPs are abundant in testis and also expressed in brain, pituitary, eye, and other mouse tissues. In brain, Nnal/CCPl, CCP5, and CCP6 are broadly distributed, whereas CCP2 and 3 exhibit restricted patterns of expression. Nnal/CCPl, CCP2, CCP5, and CCP6 were found to exhibit a cytosolic distribution, with a slight accumulation of CCP5 in the nucleus. Based on the above results, we hypothesized that Nnal/CCPl and CCP2‐6 function in the processing of cytosolic proteins such as alpha‐tubuHn, which is known to be modified by the removal of a C‐terminal tyrosine. Analysis of the forms of alpha tubulin in the olfactory bulb of mice lacking Nnal/CCPl showed the absence of the detyrosinylated form in the mitral cells. Taken together, these results are consistent with a role for Nnal/CCPl and the related CCPs in the processing of tubulin.—Kalinina, E., Biswas, R., Berezniuk, I., Hermoso, A., Aviles, F. X., Fricker, L. D. A novel subfamily of mouse cytosolic carboxypeptidases. FASEB J. 21, 836–850 (2007)

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel β-sheet and one short α-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

New palladium(II) and platinum(II) complexes with 9-aminoacridine: structures, luminiscence, theoretical calculations, and antitumor activity.

The new complexes [Pd(dmba)( N10-9AA)(PPh 3)]ClO 4 ( 1), [Pt(dmba)( N9-9AA)(PPh 3)]ClO 4 ( 2), [Pd(dmba)( N10-9AA)Cl] ( 3), and [Pd(C 6F 5)( N10-9AA)(PPh 3)Cl] ( 4) (9-AA = 9-aminoacridine; dmba = N,C-chelating 2-(dimethylaminomethyl)phenyl) have been prepared. The crystal structures have been established by X-ray diffraction. In complex 2, an anagostic C-H...Pt interaction is observed. All complexes are luminescent in the solid state at room temperature, showing important differences between the palladium and platinum complexes. Complex 2 shows two structured emission bands at high and low energies in the solid state, and the lifetimes are in agreement with excited states of triplet parentage. Density functional theory and time-dependent density functional theory calculations for complex 2 have been done. Values of IC 50 were also calculated for the new complexes 1- 4 against the tumor cell line HL-60. All of the new complexes were more active than cisplatin (up to 30-fold in some cases). The DNA adduct formation of the new complexes synthesized was followed by circular dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the complexes on plasmid DNA pB R322 were also obtained.

Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily

Nnal has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nnal has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nnal homologues in bacteria, Protista, and Anima‐lia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nnal‐like sequences demonstrates a major divergence between Nnal‐like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational mod‐eling of representative Nnal‐like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nnal‐like peptidases from Caenσrhabditis elegans and demonstrated that this protein is a fully functional metaUocarboxypeptidase that cleaves a range of C‐terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt‐inactivated, and is preferentially inhibited by Z‐Glu‐Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal (1). Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nnal‐like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.—Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., Avilés, F. X. Nna1‐like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily. FASEB J. 20, 851–865 (2007)

Complete amino acid analysis of proteins by dabsyl derivatization and reversed-phase liquid chromatogrphy

Abstract A method is described for the quantification of the primary amino acids in protein hydrolysates by dabsylation and high-performance liquid chromatography. Improvements in the established conditions for the formation and storage of amino acid dabsyl derivatives and the use of new reversed-phase columns allow the chromatographic analysis in 25 min of all the proteinogenic amino acids in well resolved peaks of homogenous and highly reproducible size. The method permits the simultaneous quantification of tryptophan residues by previous hydrolysis of the protein with sulphonic acids. The other acid-labile residues, asparagine and glutamine, can also be analysed by previous conversion into diaminopropionic and diaminobutyric acids, respecitvely, by treatment of the protein with [bis(trifluoroacetoxyiodo]-benzene. An extended chromatographic gradient programme allows the separation of many modified amino acids, naturally occurring or produced after chemical modification of proteins. The above characteristics together with a demonstrated high reproducibility (relative standard deviation 2.1%), flexibility, sensitivity (below 100 pmol), and inertness to extraneous chromatographic contamination make this improved method a good alternative to other currently used chromatographic methods for amino acid analysis.