RICERCA ITALIANA     

Research program

Role of metals – Ubiquitin/Proteasome interaction in the pathogenesis of conformational diseases

University Co-ordinator

Seconda Università degli Studi di NAPOLI - SCIENZE AMBIENTALI - ()

Research Unit Leader

Benedetto Di Blasio

Description

This research program has the aim to give a contribution to elucidate the mechanism of metal ions UPS inhibition focusing especially on the zinc ion. In particular, we will study the zinc-ubiquitin interaction and define the role of the metal in modulating the recognition of the ubiquitin proteolytic signal.

During this research project, we intend to fulfil the following goals:

1) NMR structural characterization of the interaction of Zn(II) with monoubiquitin;
2) Evaluation of the effect of the metal ion on the interaction of monoubiquitin with the two UBA domains of human hHR23a and the two UIM domains of the human proteasome subunit S5a;
3) NMR structural characterization of the interaction of zinc ion with a ubiquitin Lys48 linked dimer (K48Ub2);
4) NMR structural characterization of the interaction of K48Ub2 with the UBA domains of human hHR23A and the UIM domains of the human proteasome subunit S5a;
5) Evaluation of the effect of the metal ion on the interaction of K48Ub2 dimer with the UBA domains of human hHR23A and the UIM domains of the human proteasome subunit S5a.

1) NMR structural characterization of the interaction of Zn(II) with monoubiquitin;

To gain insight into the molecular basis of the interaction of Zn(II) with monoubiquitin we will conduct NMR chemical shift perturbation studies. Titration of 15N-labeled monoubiquitin with zinc ion will be conducted, by acquiring a series of 1H-15N HSQC of ubiquitin with increasing amounts of Zn(II). Changes of 15N and 1H chemical shifts upon Zn(II) addition will be quantified and the amino acid residues involved in ubiquitin-metal ion interaction will be mapped on the three-dimensional structure of the protein. An estimation of the binding constant will be also achieved and the structure of the ubiquitin-Zn(II) complex will be obtained by acquiring a set of 3D 15N [1H-1H]-NOESY, 3D 15N [1H-1H]-TOCSY, 3D-HNHA, 3DHNHB, 2D NOESY, TOCSY and DQFCOSY spectra and using the derived distance and dihedral constraints for structure calculation.

2) Evaluation of the effect of the metal ion on the interaction of monoubiquitin with the two UBA domains of human hHR23A and the two UIM domains of the human proteasome subunit S5a;

Ubiquitin has been shown to interact with both UBA domains present in the hHR23A proteins (UBA1 and UBA2) and with the two UIM domains of the human proteasome subunit S5a (UIM1 and UIM2). These interactions are believed to be essential for recognition of the ubiquitinated proteins by the proteasome machinery for their degradation. In order to assess if the interaction of ubiquitin with the zinc ion would interfere with its binding properties, we will perform NMR studies to verify the interaction of monoubiquitin with the UBAs and UIMs domains in the presence of zinc. A series of 1H-15N HSQC of 15N-labeled monoubiquitin will be acquired with increasing amounts of unlabeled hHR23A-UBA1, hHR23A-UBA2, S5a-UIM1 and S5a-UIM2, either in the absence and in presence of zinc and the differences will be analysed.

3) NMR structural characterization of the interaction of zinc ion with a ubiquitin Lys48 linked dimer (K48Ub2).

Because Lys48 linked polyubiquitin chains modulates Ub-mediated proteolytic signaling, we will be interested in determining the interaction of metals with polyubiquitin. As a model system of a ubiquitin polymer we will use a Lys 48-linked Ub2 dimer (K48Ub2). To overcome the expected spectroscopic equivalence of the two monomers in the homopolymeric chain, we will prepare for NMR analysis dimers in which only one Ub unit will be isotope-enriched. The segmentally isotope-enriched K48Ub2 will be produced according to the protocol described by Varadan et al (25). Two different K48Ub2 dimers will be obtained, named P for proximal and D for distal (with respect to a possible substrate), depending on the location of the isotope-enriched Ub moiety in the Ub2 molecule. Titration of segmentally 15N-enriched Ub2 dimers (Ub2P and Ub2D) with Zn(II) will be conducted, acquiring a series of 1H-15N HSQC of either P and D K48Ub2 with increasing amounts of Zn(II). The chemical shift mapping of the varied 15N and 1H chemical shifts of the backbone amides of P and D dimers will be used to identify sites of the Ub2 protein interacting with the metal ion.

4) NMR structural characterization of the interaction of K48Ub2 with the UBA domains of human hHR23A and the UIM domains of the human proteasome subunit S5a;

Although monoubiquitin/UBA and monoubiquitin/UIM complexes are well characterized, much less is known about UBA/polyubiquitin and UIM/polyubiquitin complexes, even though polyubiquitin chains are believed to be the biologically relevant ligands of most UBA and UIM protein domains.
We will start to gain structural insights into protein binding properties of K48Ub2 by mapping K48Ub2-UBA and K48Ub2-UIM interactions. In particular, the K48Ub2-UBA and K48Ub2-UIM interaction surface will be mapped using chemical shift perturbations in a series of NMR titration experiments. Segmentally 15N-labeled K48Ub2 samples (Ub2P and Ub2D) will be titrated with increasing amount of unlabeled hHR23A-UBA1, hHR23A-UBA2, S5a-UIM1 and S5a-UIM2. Binding will be monitored through changes in the peak positions in a series of 1H,15N HSQC spectra with increasing relative concentrations of the binding domains.
To get more defined NMR structural informations about the contact surfaces between the binding domain and the ubiquitin dimers, we will also produce 15N–labeled and 15N 13C-labeled hHR23A-UBA2 and S5a-UIM2 samples and will use them to produce complexes with the segmentally 15N-labeled K48Ub2 samples. By acquiring a 13C-separated-12C-filtered-NOESY spectrum will obtain distance constraints to build a structural model of the protein-protein interaction.


5) Evaluation of the effect of the metal ion on the interaction of the K48Ub2 dimer with the UBA domains of human hHR23A and the UIM domains of the human proteasome subunit S5a.

In order to assess if the interaction of Ub2 with zinc ion would interfere with its binding properties, we will perform NMR studies to verify the interaction of K48Ub2 with the UBAs and UIMs domains in the presence of Zn(II). A series of 1H-15N HSQC of segmentally 15N-labeled K48Ub2 (Ub2P and Ub2D) will be acquired with increasing amounts of unlabeled hHR23A-UBA1, hHR23A-UBA2, S5a-UIM1 and S5a-UIM2, either in the absence and in presence of zinc and the differences will be analysed.


Experimental plan:

Goal 1

Phase 1.1. Cloning of the DNA coding sequence of the human ubiquitin in a suitable vector for the expression in E. Coli, expression of the 15N-labeled protein sample and purification.
Scheduled time: 0,5 month
Phase 1.2 NMR spectra acquisition at different metal ions concentration.
Scheduled time: 1 months
Phase 1.3 Structure determinations of monoubiquitin-metal complexes via NMR techniques.
Scheduled time: 3 months

Goal 2

Phase 2.1. Cloning of the DNA coding sequence of the two UBA domains present in the hHR23A proteins and the two UIM domains of the human proteasome subunit S5a in suitable vectors for the expression in E. Coli; expression of the protein samples and purification.
Scheduled time: 1,5 month
Phase 2.2 NMR spectra acquisition of the 2 UBA-monoubiquitin, and the 2 UIM-monoubiquitin complexes in the absence and in the presence of the metal.
Scheduled time: 2 months
Phase 2.3 Analysis of the NMR spectra to evaluate the effect of the zinc ion on the monoubiquitin-UIM and monoubiquitin-UBA complexes.
Scheduled time: 2 months

Goal 3

Phase 3.1. Preparation of segmentally 15N-enriched K48Ub2 samples according to Varadan’s protocol (25)
Scheduled time: 1 month
Phase 3.2 NMR spectra acquisition of the segmentally 15N-enriched K48Ub2 in the absence and in the presence of the metal.
Scheduled time: 1 months
Phase 3.3 Analysis of the spectra to evaluate the interaction between the zinc ion and the K48Ub2 dimer.
Scheduled time: 2 months


Goal 4

Phase 4.1 NMR spectra acquisition of the complexes between the segmentally 15N-enriched K48Ub2 and the hHR23A-UBA1, hHR23A-UBA2, S5a-UIM1 and S5a-UIM2 protein domains.
Scheduled time: 2 months
Phase 4.2 Analysis of the spectra to evaluate the interaction surfaces between the K48Ub2 and the different binding domains.
Scheduled time: 4 months


Goal 5

Phase 5.1 NMR spectra acquisition of the complexes between the segmentally 15N-enriched K48Ub2 and the hHR23A-UBA1, hHR23A-UBA2, S5a-UIM1 and S5a-UIM2 protein domains in the presence of the zinc ion.
Scheduled time: 2 months
Phase 5.2 Analysis of the spectra to evaluate the effect of the metal ion on the interaction between the K48Ub2 and the different binding domains.
Scheduled time: 2 months

All our experiments will be conducted in strict collaboration with the research unit in Catania to elucidate the binding site, the coordination features, and the affinity of metal ions to ubiquitin and polyubiquitin and to assess the effects of metal binding on the stability and structure of the protein. Moreover the new specific molecules able to interfere with the metal-mediated inhibition of UPS, designed by the unit in Siena, will be also tested in our systems to evaluate their effect on the metal-proteins interactions.

This study relies on the scientific results obtained by the members of our research unit in:
a) the expression, purification and functional characterization of proteins (68-70);
b) the NMR solution determination of the three-dimensional structure of small molecules, peptides and proteins (71-78).

For the NMR experiments the unity of Caserta will use, for the data collection, the NMR spectrometer, operating at 500 MHz present at the Department of Environmental Sciences of the Second University of Naples, and the NMR spectrometer operating at 600 MHz present at the Istituto di Biostrutture e Bioimmagini of the National Research Centre (CNR) of Naples, that will be available for this project. The mono- and multi-dimensional spectra elaboration and the NMR-based structure calculation will be performed using the Octane SGI work stations present in laboratories of the unit of Caserta at the Department of Enviromental Sciences. The expression of the proteins will be also performed in the laboratories of the unit of Caserta.