This article describes the general method to perform the classical two‐hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein‐protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two‐hybrid assay have been proven to be binary direct interactions. Data comparison with other systems, such as mass spectrometry, have demonstrated that this system is at least as reliable. In fact, its use is increasing with time, and at present numerous variants of the yeast two‐hybrid assay have been developed, including high‐throughput systems that promote the generation of a proteome‐scale map of protein‐protein interactions in specific system. © 2018 by John Wiley & Sons, Inc.

Neurodegenerative diseases are pathologies of the central and peripheral nervous systems characterized by loss of brain functions and problems in movement which occur due to the slow and progressive degeneration of cellular elements. Several neurodegenerative diseases are known such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis and many studies on the molecular mechanisms underlying these pathologies have been conducted. Altered functions of some key proteins and the presence of intraneuronal aggregates have been identified as responsible for the development of the diseases. Interestingly, the formation of the SNARE complex has been discovered to be fundamental for vesicle fusion, vesicle recycling and neurotransmitter release. Indeed, inhibition of the formation of the SNARE complex, defects in the SNARE-dependent exocytosis and altered regulation of SNARE-mediated vesicle fusion have been associated with neurodegeneration. In this review, the biological aspects of neurodegenerative diseases and the role of SNARE proteins in relation to the onset of these pathologies are described.

Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway.

The small GTPases of the Rho family regulate many aspects of actin dynamics, but are functionally connected to many other cellular processes. Rac1, a member of this family, besides its known function in the regulation of actin cytoskeleton, plays a key role in the production of reactive oxygen species, in gene transcription, in DNA repair, and also has been proven to have specific roles in neurons. This review focuses on the cooperation between Rac1 and Rab proteins, analyzing how the coordination between these GTPases impact on cells and how alterations of their functions lead to disease.

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion.