Machine-learning methods for structure prediction of β-barrel membrane proteins

Different types of proteins exist with diverse functions that are essential for living organisms. An important class of proteins is represented by transmembrane proteins which are specifically designed to be inserted into biological membranes and devised to perform very important functions in the cell such as cell communication and active transport across the membrane. Transmembrane β-barrels (TMBBs) are a sub-class of membrane proteins largely under-represented in structure databases because of the extreme difficulty in experimental structure determination. For this reason, computational tools that are able to predict the structure of TMBBs are needed. In this thesis, two computational problems related to TMBBs were addressed: the detection of TMBBs in large datasets of proteins and the prediction of the topology of TMBB proteins. Firstly, a method for TMBB detection was presented based on a novel neural network framework for variable-length sequence classification. The proposed approach was validated on a non-redundant dataset of proteins. Furthermore, we carried-out genome-wide detection using the entire Escherichia coli proteome. In both experiments, the method significantly outperformed other existing state-of-the-art approaches, reaching very high PPV (92%) and MCC (0.82). Secondly, a method was also introduced for TMBB topology prediction. The proposed approach is based on grammatical modelling and probabilistic discriminative models for sequence data labeling. The method was evaluated using a newly generated dataset of 38 TMBB proteins obtained from high-resolution data in the PDB. Results have shown that the model is able to correctly predict topologies of 25 out of 38 protein chains in the dataset. When tested on previously released datasets, the performances of the proposed approach were measured as comparable or superior to the current state-of-the-art of TMBB topology prediction.

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

From "wet biology" to statistical analysis of structural features with bioinformatics tools

Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five—YftM, YaiO, YfaZ, CsgF, and YliI—and also provide preliminary data indicating that a sixth—YfaL—may be an outer membrane autotransporter.

Use of E. coli OMVs as delivery system to elicit neutralizing antibodies against Chlamydia infectivity: the HtrA protein example

The obligate intracellular pathogen Chlamydia trachomatis is a gram negative bacterium which infects epithelial cells of the reproductive tract. C. trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and a vaccine against this pathogen is highly needed. Many evidences suggest that both antigen specific-Th1 cells and antibodies may be important to provide protection against Chlamydia infection. In a previous study we have identified eight new Chlamydia antigens inducing CD4-Th1 and/or antibody responses that, when combined properly, can protect mice from Chlamydia infection. However, all selected recombinant antigens, upon immunization in mice, elicited antibodies not able to neutralize Chlamydia infectivity in vitro. With the aim to improve the quality of the immune response by inducing effective neutralizing antibodies, we used a novel delivery system based on the unique capacity of E. coli Outer Membrane Vesicles (OMV) to present membrane proteins in their natural composition and conformation. We have expressed Chlamydia antigens, previously identified as vaccine candidates, in the OMV system. Among all OMV preparations, the one expressing HtrA Chlamydia antigen (OMV-HtrA), showed to be the best in terms of yield and quantity of expressed protein, was used to produce mice immune sera to be tested in neutralization assay in vitro. We observed that OMV-HtrA elicited specific antibodies able to neutralize efficiently Chlamydia infection in vitro, indicating that the presentation of the antigens in their natural conformation is crucial to induce an effective immune response. This is one of the first examples in which antibodies directed against a new Chlamydia antigen, other than MOMP (the only so far known antigen inducing neutralizing antibodies), are able to block the Chlamydia infectivity in vitro. Finally, by performing an epitope mapping study, we investigated the specificity of the antibody response induced by the recombinant HtrA and by OMV-HtrA. In particular, we identified some linear epitopes exclusively recognized by antibodies raised with the OMV-HtrA system, detecting in this manner the antigen regions likely responsible of the neutralizing effect.