Lorenza Trabalzini

Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae

We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.

Identification of a Novel RalGDS-related Protein as a Candidate Effector for Ras and Rap1

Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

RalGDS family members couple Ras to Ral signalling and that's not all.

Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.

The Ras Superfamily of Small GTPases: The Unlocked Secrets

The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes. Fuelled by the original identification in 1982 of mutationally activated and transforming human Ras genes in human cancer cell lines, a variety of powerful experimental techniques have been intensively focused on discovering and studying structure, biochemistry, and biology of Ras and Ras-related small GTPases, leading to fundamental research breakthroughs into identification and structural and functional characterization of a huge number of Ras superfamily members, as well as of their multiple regulators and effectors. In this review we provide a general overview of the major milestones that eventually allowed to unlock the secret treasure chest of this large and important superfamily of proteins.

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

Molecular Crosstalk between Integrins and Cadherins: Do Reactive Oxygen Species Set the Talk?

The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis. However, the molecular mechanisms underlying the fine-tuned functional communication between cadherins and integrins are still elusive. This paper focuses on recent findings towards the involvement of reactive oxygen species (ROS) in the regulation of cell adhesion and signal transduction functions of integrins and cadherins, pointing to ROS as emerging strong candidates for modulating the molecular crosstalk between cell-matrix and cell-cell adhesion receptors.

Defective autophagy is a key feature of cerebral cavernous malformations

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss‐of‐function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss‐of‐function activates the mTOR‐ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole‐cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2‐silenced human endothelial cells and in both cells and tissues from an endothelial‐specific CCM3‐knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial‐to‐mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

The Interplay between ROS and Ras GTPases: Physiological and Pathological Implications.

The members of the RasGTPase superfamily are involved in various signaling networks responsible for fundamental cellular processes. Their activity is determined by their guanine nucleotide-bound state. Recent evidence indicates that some of these proteins may be regulated by redox agents. Reactive oxygen species (ROSs) and reactive nitrogen species (RNSs) have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that the intracellular production of ROS is tightly regulated and that these redox agents serve as signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some Ras GTPases appear to regulate ROS production and that oxidants function as effector molecules for the small GTPases, thus contributing to their overall biological function. Thus, redox agents may act both as upstream regulators and as downstream effectors of Ras GTPases. Here we discuss current understanding concerning mechanisms and physiopathological implications of the interplay between GTPases and redox agents.

In vivo 13C-NMR and modelling study of metabolic yield response to ethanol stress in a wild-type strain of Saccharomyces cerevisiae

In this paper the combined use of in vivo 13C‐nuclear magnetic resonance spectroscopy and mathematical modelling allowed the analysis of the response to ethanol stress in a wild‐type strain of Saccharomyces cerevisiae, in terms of a reduced metabolic activity. The model developed succeeded in describing and interpreting the effects of increasing concentrations of exogenous ethanol. In particular, the ratio between the kinetic constants associated with ethanol production and glucose consumption gave the estimation of the metabolic yield of the processes in perfect agreement with experimental results.

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.