Leonardo G. Alonso

UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates.

Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexin/calreticulin, CNX/CRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. Structural characterization of species showing higher glucose acceptor capacity suggests that GT recognizes solvent accessible hydrophobic patches in molten globule-like conformers mimicking intermediate folding stages of nascent glycoproteins. It was further confirmed that BiP (binding protein, a chaperone of the heat shock protein 70 family) preferentially recognized neoglycoproteins displaying extended conformations, thus providing a molecular rationale for the sequential BiP-CNX/CRT interaction with folding glycoproteins observed in vivo.

The Catalytic Domain of Insulin-degrading Enzyme Forms a Denaturant-resistant Complex with Amyloid β Peptide IMPLICATIONS FOR ALZHEIMER DISEASE PATHOGENESIS

Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta (Abeta) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type 2. Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Abeta that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in an SDS-soluble fraction from AD cortical tissue. Abeta sequence 17-27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Abeta was competent to associate irreversibly with IDE following a very slow kinetics (t(1/2) approximately 45 min). Partial denaturation of IDE as well as preincubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Abeta takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Abeta remained bound to a approximately 25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE .Abeta complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Abeta. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Abeta clearance, insulin turnover, and promote AD pathogenesis.Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid β (Aβ) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type 2. Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Aβ that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in an SDS-soluble fraction from AD cortical tissue. Aβ sequence 17–27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Aβ was competent to associate irreversibly with IDE following a very slow kinetics (t½ ∼ 45 min). Partial denaturation of IDE as well as preincubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Aβ takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Aβ remained bound to a ∼25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE ·Aβ complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Aβ. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Aβ clearance, insulin turnover, and promote AD pathogenesis.

SPARC modulates the proliferation of stromal but not melanoma cells unless endogenous SPARC expression is downregulated

Cell interaction with the extracellular matrix (ECM) has profound influence in cancer progression. The secreted protein, acidic and rich in cysteine (SPARC) a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. This apparent paradox might result either from the biochemical properties of the different SPARC sources or from differential responses of malignant and stromal cells to SPARC. To test these hypotheses, we purified SPARC secreted by melanoma cells (hMel‐SPARC) and compared its activity with different recombinant SPARC preparations, including a new one produced in insect cells. All 5 SPARC species were effective in inhibiting bovine aortic endothelial cell proliferation, adhesion and migration. We then used the melanoma‐derived protein to assess SPARC effect on additional cell types. hMel‐SPARC greatly impaired the proliferation of both normal and transformed human endothelial cells and exerted a moderate biphasic effect on human fetal fibroblasts proliferation, irrespective of their endogenous SPARC levels. However, SPARC had no effect on the proliferation of several human cancer cell lines regardless of their endogenous levels of SPARC expression. Importantly, downregulation of SPARC levels in melanoma cells using either an antisense RNA or a shRNA against SPARC sensitized them to hMel‐SPARC addition in proliferation and migration assays, suggesting that malignant cells developed a SPARC‐resistance mechanism. This was not a general resistance to growth suppressing agents, as melanoma cells with restricted SPARC expression were more resistant to chemotherapeutic agents. Thus, malignant cells expressing or not expressing SPARC developed alternative mechanisms that, in contrary to stromal cells, rendered them SPARC‐insensitive.

Sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein.

In the present work, we have used the papillomavirus E7 oncoprotein to pursue structure-function and evolutionary studies that take into account intrinsic disorder and the conformational diversity of globular domains. The intrinsically disordered (E7N) and globular (E7C) domains of E7 show similar degrees of conservation and co-evolution. We found that E7N can be described in terms of conserved and coevolving linear motifs separated by variable linkers, while sequence evolution of E7C is compatible with the known homodimeric structure yet suggests other activities for the domain. Within E7N, inter-residue relationships such as residue co-evolution and restricted intermotif distances map functional coupling and co-occurrence of linear motifs that evolve in a coordinate manner. Within E7C, additional cysteine residues proximal to the zinc-binding site may allow redox regulation of E7 function. Moreover, we describe a conserved binding site for disordered domains on the surface of E7C and suggest a putative target linear motif. Both homodimerization and peptide binding activities of E7C are also present in the distantly related host PHD domains, showing that these two proteins share not only structural homology but also functional similarities, and strengthening the view that they evolved from a common ancestor. Finally, we integrate the multiple activities and conformations of E7 into a hierarchy of structure-function relationships.

Circular Dichroism Techniques for the Analysis of Intrinsically Disordered Proteins and Domains

Circular dichroism (CD) spectroscopy is a simple and powerful technique, which allows for the assessment of the conformational properties of a protein or protein domain. Intrinsically disordered proteins (IDPs), as discussed throughout this series, differ from random coil polypeptides in that different regions present specific conformational preferences, exhibiting dynamic secondary structure content [1]. These dynamic secondary structure elements can be stabilized or perturbed by different chemical (solvent, ionic strength, pH) or physical (temperature) agents, by posttranslational modifications, and by ligands. This information is important for defining ID nature. As IDPs present dynamic conformations, circular dichroism measurements (and other approaches as well) should be carried out not as single spectra performed in unique conditions, but instead changing the chemical conditions and observing the behavior, as part of the determination of the ID nature.In this chapter, we present the basic methodology for performing Far-UV CD measurements on a protein of interest and for identifying and characterizing intrinsically disordered regions, and several protocols for the analysis of residual secondary structure present in the protein under study. These techniques are straightforward to perform; they require minimal training and can be preliminary to more complex methodologies such as NMR.

The Human Papillomavirus E7−E2 Interaction Mechanism in Vitro Reveals a Finely Tuned System for Modulating Available E7 and E2 Proteins

Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a >or=2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a K(D) of 0.1 muM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.

Amino Acid Metabolism Conflicts with Protein Diversity

The 20 protein-coding amino acids are found in proteomes with different relative abundances. The most abundant amino acid, leucine, is nearly an order of magnitude more prevalent than the least abundant amino acid, cysteine. Amino acid metabolic costs differ similarly, constraining their incorporation into proteins. On the other hand, a diverse set of protein sequences is necessary to build functional proteomes. Here, we present a simple model for a cost-diversity trade-off postulating that natural proteomes minimize amino acid metabolic flux while maximizing sequence entropy. The model explains the relative abundances of amino acids across a diverse set of proteomes. We found that the data are remarkably well explained when the cost function accounts for amino acid chemical decay. More than 100 organisms reach comparable solutions to the trade-off by different combinations of proteome cost and sequence diversity. Quantifying the interplay between proteome size and entropy shows that proteomes can get optimally large and diverse.

Cysteine-rich positions outside the structural zinc motif of human papillomavirus E7 provide conformational modulation and suggest functional redox roles

The E7 protein from high-risk human papillomavirus is essential for cell transformation in cervical, oropharyngeal, and other HPV-related cancers, mainly through the inactivation of the retinoblastoma (Rb) tumor suppressor. Its high cysteine content (~7%) and the observation that HPV-transformed cells are under oxidative stress prompted us to investigate the redox properties of the HPV16 E7 protein under biologically compatible oxidative conditions. The seven cysteines in HPV16 E7 remain reduced in conditions resembling the basal reduced state of a cell. However, under oxidative stress, a stable disulfide bridge forms between cysteines 59 and 68. Residue 59 has a protective effect on the other cysteines, and its mutation leads to an overall increase in the oxidation propensity of E7, including cysteine 24 central to the Rb binding motif. Gluthationylation of Cys 24 abolishes Rb binding, which is reversibly recovered upon reduction. Cysteines 59 and 68 are located 18.6 Å apart, and the formation of the disulfide bridge leads to a large structural rearrangement while retaining strong Zn association. These conformational and covalent changes are fully reversible upon restoration of the reductive environment. In addition, this is the first evidence of an interaction between the N-terminal intrinsically disordered and the C-terminal globular domains, known to be highly and separately conserved among human papillomaviruses. The significant conservation of such noncanonical cysteines in HPV E7 proteins leads us to propose a functional redox activity. Such an activity adds to the previously discovered chaperone activity of E7 and supports the picture of a moonlighting pathological role of this paradigmatic viral oncoprotein.

Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain

Background Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. Methodology/Principal Findings Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric “Z-nucleus” after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a “conformation editing” mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. Conclusion We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions.

Long-lasting immunoprotective and therapeutic effects of a hyperstable E7 oligomer based vaccine in a murine human papillomavirus tumor model†

Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high‐risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV‐induced cancer cells. We have previously shown that E7 can self‐assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV‐tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG‐rich oligonucleotide (ODN) as adjuvant elicits a strong long‐lasting protection against E7‐expressing tumor cells, preventing tumor outgrowth after rechallenge 90‐days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose‐dependent manner. These proof‐of‐principle preclinical experiments denote the potential applicability of our E7SOx‐based vaccine to the treatment of cervical cancer and other mucosal HPV‐related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low‐cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age.