RICERCA ITALIANA     

Protein interactomes: unravelling cellular networks in different pathophysiological conditions

Università degli Studi di Napoli "Federico II"

Abstract

Cellular systems depend on multiprotein complexes in which individual proteins assemble into functional modules. Most gene products mediate their function within complex networks of interconnected macromolecules, which have topological and dynamic properties that reflect biological phenomena. Thus, an understanding of biological mechanisms and disease processes demands global analyses of the structure, function and dynamics of the complex networks in which macromolecules function.
The aim of this project is to identify, characterize and functionally describe, using biochemical, molecular biology and proteomic-derived technologies, macromolecular interaction networks of three proteins, which are involved in different physiopathological pathways, to generate mechanistic models that will be implemented into a bioinformatics platform. Each research Units will contribute to the project with their specific know-how and facilities.
The Units I of Prof. Salvatore and the Unit II of Prof Orrù will study the function of two proteins: 1) the putative transcription factor AF4, which is involved in the pathogenesis of human acute lymphoblastic leukemia; and 2) the brain specific glycolytic enzyme, aldolase C, recently reported as a moonlight activity protein with prion-protein binding properties. Both proteins are also involved in the pathogenesis of neurodegenerative diseases.
Unit I will use interaction-detection methods (two-hybrid system, affinity >>>

Principal Investigator

Francesco Salvatore Università degli Studi di NAPOLI "Federico II"

Research Objectives

The aim of this project is to identify, characterize and functionally describe, using proteomic-derived technologies, macromolecular interaction networks of three proteins, which are involved in different physiological and pathological pathways, to generate mechanistic models that will be implemented into a bioinformatics platform.
Using high-throughput interaction-detection approaches, namely, yeast two-hybrid systems, affinity co-immunoprecipitation, GST pull-down, functional and comparative proteomic analysis, and bioinformatics tools we will determine the function of: 1) the putative transcription factor AF4 which is involved in the pathogenesis of human leukemia and neurodegenerative diseases; 2) the brain specific glycolytic enzyme, aldolase C, recently reported as a “moonlight” activity protein with prion-protein binding properties; and 3) immunoglobulin light chains in systemic amyloidosis pathogenesis.
Each research unit has specific know-how and facilities related to the issues addressed in the project.
A common feature of AF4 and aldolase C is that they are also specifically expressed in Purkinje cells in the brain. AF4 is mutated in the “robotic mouse”, a novel model of autosomal dominant cerebellar ataxia. The mutation, which lies in a highly conserved region in members of the ALF family, significantly reduces the binding affinity of AF4 to the E3 ubiquitin-ligase Siah-1a, which has been isolated with Siah-2 as interacting proteins in the >>>

First Results

FUNCTIONAL PROTEOMICS OF AF4. The main expected results of this study are:
- identification of AF4 C-terminal domain interactome;
- isolation of the transcriptional regulatory complex containing AF4;
- identification of AF4 target genes, whose altered regulation might provoke either leukemia or neurodegeneration.
- identification of direct interactors that could give information about AF4 function and regulation pathways.
Our experiments will identify potential AF4 partners and bioinformatics will verify if they belong to the transcriptional regulatory complex that contains AF4.
Indeed, we already identified a series of AF4 molecular partners using as bait the N-terminal peptide, which spans the transactivation domain. We identified p-TEFb kinase and 14-3-3 theta as AF4 interactors. 14-3-3 proteins, involved in human cancer and neurological disorders, regulate many cellular processes by binding to phosphorylated sites in diverse target proteins. 14-3-3s themselves are phosphorylated. It is known that phosphorylation regulates the AF4 function (14). Therefore, 14-3-3 binding may variously govern AF4 regulation by i) inducing conformational changes, (ii) physically occluding sequence-specific or structural features, (iii) scaffolding, and (iv) changing cellular localization.
To assess the function of the putative direct interaction between AF4 and 14-3-3 theta, bioinformatics study of single domains of the large AF4 protein >>>

Timescale

24 months

National and international background

Cellular systems depend on multi-protein complexes in which individual proteins assemble into functional modules (1,2). Most gene products mediate their function within complex networks of interconnected macromolecules, which have topological and dynamic properties that reflect biological mechanisms as well as disease processes. (3,4).
To understand the interaction network in which macromolecules function, high-throughput interaction detection approaches, such as yeast two-hybrid systems (5,6), proteomic analysis (7,8), and ‘in silico' interaction predictions (9-14), have been developed. In particular, proteomic analysis may potentially look for all the major proteins involved in specific pathways and identify differences in protein expression patterns in different cellular states.
To obtain information about biological activities of individual proteins, “functional proteomics” analyzes spatial and temporal properties of molecular networks and fluxes in living cells. In the cell, many processes are governed not only by the relative abundance of proteins but also by rapid and transient regulation of the activity, association and localization of proteins and protein complexes. Proteomics-based approaches were crucial in identifying interacting proteins in large, stable complexes (15). The simplest strategy to identify in vivo proteins that, even transiently, interact with a chosen protein target entails the use of commercially available protein expression >>>