Josep Vendrell

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.

Design, selection, and characterization of thioflavin-based intercalation compounds with metal chelating properties for application in Alzheimer's disease.

Metal chelation is considered a rational therapeutic approach for interdicting Alzheimers amyloid pathogenesis. At present, enhancing the targeting and efficacy of metal-ion chelating agents through ligand design is a main strategy in the development of the next generation of metal chelators. Inspired by the traditional dye Thioflavin-T, we have designed new multifunctional molecules that contain both amyloid binding and metal chelating properties. In silico techniques have enabled us to identify commercial compounds that enclose the designed molecular framework (M1), include potential antioxidant properties, facilitate the formation of iodine-labeled derivatives, and can be permeable through the blood-brain barrier. Iodination reactions of the selected compounds, 2-(2-hydroxyphenyl)benzoxazole (HBX), 2-(2-hydroxyphenyl)benzothiazole (HBT), and 2-(2-aminophenyl)-1H-benzimidazole (BM), have led to the corresponding iodinated derivatives HBXI, HBTI, and BMI, which have been characterized by X-ray diffraction. The chelating properties of the latter compounds toward Cu(II) and Zn(II) have been examined in the solid phase and in solution. The acidity constants of HBXI, HBTI, and BMI and the formation constants of the corresponding ML and ML2 complexes [M = Cu(II), Zn(II)] have been determined by UV-vis pH titrations. The calculated values for the overall formation constants for the ML2 complexes indicate the suitability of the HBXI, HBTI, and BMI ligands for sequestering Cu(II) and Zn(II) metal ions present in freshly prepared solutions of beta-amyloid (Abeta) peptide. This was confirmed by Abeta aggregation studies showing that these compounds are able to arrest the metal-promoted increase in amyloid fibril buildup. The fluorescence features of HBX, HBT, BM, and the corresponding iodinated derivatives, together with fluorescence microscopy studies on two types of pregrown fibrils, have shown that HBX and HBT compounds could behave as potential markers for the presence of amyloid fibrils, whereas HBXI and HBTI may be especially suitable for radioisotopic detection of Abeta deposits. Taken together, the results reported in this work show the potential of new multifunctional thioflavin-based chelating agents as Alzheimers disease therapeutics.

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.

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.

Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program

OBJECTIVE To evaluate the frequency of disomy (for chromosomes X, Y, and 18) and of diploidy in the spermatozoa of infertile men undergoing intracytoplasmic sperm injection (ICSI). DESIGN Prospective analysis of sperm nuclei by fluorescence in situ hybridization (FISH). SETTING University-affiliated IVF-ICSI program. PATIENT(S) Semen samples from 19 patients participating in an IVF-ICSI program. INTERVENTION(S) Semen samples were analyzed and prepared for FISH. MAIN OUTCOME MEASURE(S) Semen parameters were evaluated. The frequency of disomy for chromosomes X, Y, and 18 and the frequency of diploidy were analyzed by FISH. RESULT(S) A total of 9,373 spermatozoa from 19 infertile patients were analyzed and compared with spermatozoa from a control group of 5 healthy men. No differences in the frequency of disomy 18 were found, but statistically significant differences in the incidence of sex chromosome disomy and of diploidy were observed. CONCLUSION(S) The study of sperm nuclei by FISH is useful to improve genetic counseling in infertile patients selected for ICSI.

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.

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.

Crystal Structure of Avian Carboxypeptidase D Domain II : A Prototype for the Regulatory Metallocarboxypeptidase Subfamily

The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N‐terminal α/β‐hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel‐like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This α/β‐hydrolase subdomain is followed by a C‐terminal 80 residue β‐sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar‐binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.

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.

Metallocarboxypeptidases: Emerging drug targets in biomedicine

Metallocarboxypeptidases (MCPs) are commonly regarded as exopeptidases that actively participate in the digestion of proteins and peptides. In the recent years, however, novel MCPs comprising a wide range of physiological roles have been found in different mammalian extra-pancreatic tissues and fluids. Among them, CPU, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), has been shown to cleave C-terminal Lys residues from partially degraded fibrin, acting as inhibitor of clot fibrinolysis and therefore constituting an important drug target for thrombolytic therapies. Other MCPs such as CPE, CPN, CPM, and CPD function as pro-hormone and neuropeptide processors and display several structural differences with the pancreatic-like enzymes. In addition, important advances have been made in the discovery and characterization of new endogenous and exogenous proteinaceous inhibitors; the structural determination of their complexes with several MCPs has revealed novel binding modes. Finally, the use of MCPs in antibody-directed enzyme pro-drug therapy (ADEPT) has proved to be an efficient approach for the delivery of lethal levels of chemotherapeutic drugs specifically at tumor tissues. Taken together, these recent developments may help to understand potential biomedical implications of MCPs. Future perspectives for the regulation of these enzymes through the use of more selective and potent inhibitors are also discussed in this review and combined with earlier observations in the field.