Genotype & phenotype in Lowe Syndrome: specific OCRL1 patient mutations differentially impact cellular phenotypes.

Lowe Syndrome (LS) is a lethal genetic disorder caused by mutations in the OCRL1 gene which encodes the lipid 5' phosphatase Ocrl1. Patients exhibit a characteristic triad of symptoms including eye, brain and kidney abnormalities with renal failure as the most common cause of premature death. Over 200 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. Despite observations of heterogeneity in patient symptom severity, there is little understanding of the correlation between genotype and its impact on phenotype. Here, we show that different mutations had diverse effects on protein localization and on triggering LS cellular phenotypes. In addition, some mutations affecting specific domains imparted unique characteristics to the resulting mutated protein. We also propose that certain mutations conformationally affect the 5'-phosphatase domain of the protein, resulting in loss of enzymatic activity and causing common and specific phenotypes (a conformational disease scenario). This study is the first to show the differential effect of patient 5'-phosphatase mutations on cellular phenotypes and introduces a conformational disease component in LS. This work provides a framework that explains symptom heterogeneity and can help stratify patients as well as to produce a more accurate prognosis depending on the nature and location of the mutation within the OCRL1 gene.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization.

It is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

Lowe syndrome patient cells display mTOR- and RhoGTPase-dependent phenotypes alleviated by rapamycin and statins.

Lowe syndrome (LS) is an X-linked developmental disease characterized by cognitive deficiencies, bilateral congenital cataracts and renal dysfunction. Unfortunately, this disease leads to the early death of affected children often due to kidney failure. Although this condition was first described in the early 1950s and the affected gene (OCRL1) was identified in the early 1990s, its pathophysiological mechanism is not fully understood and there is no LS-specific cure available to patients. Here we report two important signaling pathways affected in LS patient cells. While RhoGTPase signaling abnormalities led to adhesion and spreading defects as compared to normal controls, PI3K/mTOR hyperactivation interfered with primary cilia assembly (scenario also observed in other ciliopathies with compromised kidney function). Importantly, we identified two FDA-approved drugs able to ameliorate these phenotypes. Specifically, statins mitigated adhesion and spreading abnormalities while rapamycin facilitated ciliogenesis in LS patient cells. However, no single drug was able to alleviate both phenotypes. Based on these and other observations, we speculate that Ocrl1 has dual, independent functions supporting proper RhoGTPase and PI3K/mTOR signaling. Therefore, this study suggest that Ocrl1-deficiency leads to signaling defects likely to require combinatorial drug treatment to suppress patient phenotypes and symptoms.

The Information Content of Glutamine-Rich Sequences Define Protein Functional Characteristics.

The presence of abnormally expanded glutamine (Q) repeats within specific proteins (e.g., huntingtin) are the well-established cause of several neurogenerative diseases, including Huntington disease and spinocerebellar ataxias. However, the impact of "expanded Q" stretches on the protein function is not well-understood, mostly due to lack of knowledge about the physiological role of Q repeats and the mechanism by which these repeats achieve functional-specificity. Indeed, is intriguing that regions with such low complexity (low information content) can display exquisite functional specificity. Prompting the question: where is this information stored? Applying biochemical/structural constraints and statistical analysis of protein composition we identified Q-rich (QR) regions present in coiled coils of yeast transcription factors and endocytic proteins. Our analysis indicated the existence of non-Q amino-acids differentially enriched or excluded from QR regions in one protein group versus the other. Importantly, when the non-Q amino-acids from an endocytic protein were exchanged by the ones enriched in QR from transcription factors, the resulting protein was unable to localize to the plasma membrane and was instead found in the nucleus. These results indicate that while QR repeats can efficiently engage in binding, the non-Q amino-acids provide essential specificity information. We speculate that coupling low complexity regions with information-intensive determinants might be a strategy used in many protein systems involved in different biological processes.

Role of Ocrl1 in primary cilia assembly.

Lowe syndrome is a lethal X-linked genetic disorder characterized by congenital cataracts, mental retardation, and kidney dysfunction. It is caused by mutations in the OCRL1 (oculocerebrorenal syndrome of Lowe) gene that encodes a phosphatidylinositol 5-phosphatase (EC 3.1.3.36). The gene product Ocrl1 has been linked to a multitude of functions due to the central role played by phosphoinositides in signaling. Moreover, this protein also has the ability to bind Rho GTPases, the master regulators of the actin cytoskeleton, and to interact with elements of the vesicle trafficking machinery. It is currently under investigation how deficiencies in Ocrl1 affect these different processes and contribute to patient symptoms. This chapter outlines the known physiological roles of Ocrl1 which might be relevant to the mechanism underlying Lowe syndrome.

Introduction to the analysis of the intracellular sorting information in protein sequences: from molecular biology to artificial neural networks.

A precise spatial-temporal organization of cell components is required for basic cellular activities such as proliferation and for complex multicellular processes such as embryo development. Particularly important is the maintenance and control of the cellular distribution of proteins, as these components fulfill crucial structural and catalytic functions. Membrane protein localization within the cell is determined and maintained by intracellular elements known as adaptors that interpret sorting information encoded in the amino acid sequence of cargoes. Understanding the sorting sequence code of cargo proteins would have a profound impact on many areas of the life sciences. For example, it would shed light onto the molecular mechanisms of several genetic diseases and would eventually allow us to control the fate of proteins. This chapter constitutes a primer on protein-sorting information analysis and localization/trafficking prediction. We provide the rationale for and a discussion of a simple basic protocol for protein sequence dissection looking for sorting signals, from simple sequence inspection techniques to more sophisticated artificial neural networks analysis of sorting signal recognition data.

RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking.

Cell polarity, the asymmetric distribution of proteins and lipids, is essential for a variety of cellular functions. One mechanism orchestrating cell polarity is polarized vesicle trafficking; whereby cargo loaded secretory vesicles are specifically transported to predetermined areas of the cell. The evolutionarily conserved exocyst complex and its small GTPase regulators play crucial roles in spatiotemporal control of polarized vesicle trafficking. In studies on neuronal membrane remodeling and synaptic plasticity, conserved mechanisms of exocyst regulation and cargo recycling during polarized vesicle trafficking are beginning to emerge as well. Recently, our lab demonstrated that RhoGTPase-binding proteins in both yeast (Bem3) and mammals (Ocrl1) are also required for the efficient traffic of secretory vesicles to sites of polarized growth and signaling. Together with our studies, we highlight the evolutionary conservation of the basic elements essential for polarized vesicle traffic across different cellular functions and model systems. In conclusion, we emphasize that studies on RhoGTPase-binding proteins in these processes should be included in the next level of investigation, for a more complete understanding of their hitherto unknown roles in polarized membrane traffic and exocyst regulation.

Crystallographic analysis of the ENTH domain from yeast epsin Ent2 that induces a cell division phenotype.

Epsins are eukaryotic, endocytic adaptor proteins primarily involved in the early steps of clathrin mediated endocytosis. Two epsins exist in Saccharomyces cerevisiae, Ent1 and Ent2, with single epsin knockouts being viable, while the double knockout is not. These proteins contain a highly conserved Epsin N-terminal homology (ENTH) domain that is essential for cell viability. In addition, overexpression of the ENTH domain of Ent2 (ENTH2) was shown to play a role in cell division by interacting with the septin organizing, Cdc42 GTPase activating protein, Bem3, leading to increased cytokinesis failure. In contrast, overexpression of the ENTH domain of Ent1 (ENTH1) does not affect cytokinesis, despite being 75% identical to ENTH2. An ENTH2(N112D, S114E, E118Q) mutant that switches residues in loop 7 to those found correspondingly in ENTH1 was incapable of inducing the cytokinesis phenotype. In order to better understand the role of loop 7 in the ENTH2-induced phenotype at a molecular level, X-ray crystallography was used to elucidate the structures of yeast ENTH2(WT) and ENTH2(DEQ). Our results indicate that mutations did not affect the conformation of loop 7, but rather introduce an increased negative charge on a potential interaction interface. Morphological analysis of cells overexpressing ENTH2 loop 7 mutants showed that the cytokinesis failure phenotype was abolished by the single mutants N112D, E118Q, and to a lesser extent by S114E. Taken together, our results indicate that the interaction surface that contains loop 7 and the specific nature of these residues are crucial for ENTH2 involvement in cytokinesis. This research provides insight into a molecular mechanism by which ENTH2, but not ENTH1, overexpression in yeast leads to cell division defects. Structural data of WT and mutant ENTH2 domains along with in vivo phenotypic analysis of ENTH2 overexpressing cells indicate that the biochemical nature of three loop 7 residues is crucial for its role in cytokinesis.

Bem3: Filling the GAP between cell polarity and secretion.

A highly conserved member of the Rho family of small GTPases, Cdc42 functions as the "master regulator of cell polarity." It has been reported that for proper establishment and maintenance of cell polarity, Cdc42 regulates and requires vesicle trafficking. Importantly, we recently discovered that in budding yeast, vesicle trafficking also controls the localization and function of Bem3, a GTPase activating protein for Cdc42. Specifically, we observed that Bem3 partitioned between the plasma membrane and an internal membrane-bound compartment. This Bem3-containing compartment was present during extended periods of apical growth, required actin tracks for trafficking to polarized sites and functioned as a recycling station that was positioned at the junction of endocytic and secretory pathways. Strikingly, many of these features are reminiscent of the Spitzenkörper, a dynamic structure involved in polarized growth during hyphal development in several filamentous fungi. Furthermore, Bem3 was not merely a passive cargo but actively recruited the secretory Rab GTPase Sec4 to this Spitzenkörper-like compartment. Importantly, this function of Bem3 was independent of its GAP activity. Our work demonstrates the existence of a complementary regulation between Bem3, a regulator of Cdc42 signaling and Sec4, a key component of the secretory machinery.

The epsin protein family: coordinators of endocytosis and signaling.

The epsins are a conserved family of endocytic adaptors essential for cell viability in yeast and for embryo development in higher eukaryotes. Epsins function as adaptors by recognizing ubiquitinated cargo and as endocytic accessory proteins by contributing to endocytic network stability/regulation and membrane bending. Importantly, epsins play a critical role in signaling by contributing to epidermal growth factor receptor downregulation and the activation of notch and RhoGTPase pathways. In this review, we present an overview of the epsins and emphasize their functional importance as coordinators of endocytosis and signaling.

Artificial neural network for the prediction of tyrosine-based sorting signal recognition by adaptor complexes.

Sorting of transmembrane proteins to various intracellular compartments depends on specific signals present within their cytosolic domains. Among these sorting signals, the tyrosine-based motif (YXXØ) is one of the best characterized and is recognized by µ-subunits of the four clathrin-associated adaptor complexes (AP-1 to AP-4). Despite their overlap in specificity, each µ-subunit has a distinct sequence preference dependent on the nature of the X-residues. Moreover, combinations of these residues exert cooperative or inhibitory effects towards interaction with the various APs. This complexity makes it impossible to predict a priori, the specificity of a given tyrosine-signal for a particular µ-subunit. Here, we describe the results obtained with a computational approach based on the Artificial Neural Network (ANN) paradigm that addresses the issue of tyrosine-signal specificity, enabling the prediction of YXXØ-µ interactions with accuracies over 90%. Therefore, this approach constitutes a powerful tool to help predict mechanisms of intracellular protein sorting.

The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

Fibronectin attachment protein from bacillus Calmette-Guerin as targeting agent for bladder tumor cells.

The adjuvant therapy of choice for superficial bladder cancer is the intravesical instillation of live Mycobacterium bovis bacillus Calmette-Guerin (BCG). Despite the fact that this therapy is the most effective treatment for superficial bladder cancer, intravesical administration of BCG is associated with high local morbidity and the potential for systemic infection. Therefore, there is a need for the development of safer, less toxic approaches to fight this disease. Because fibronectin attachment protein (FAP) is a key element in BCG retention and targeting to cells, we hypothesize that this protein can be used as targeting agent to deliver cytotoxic cargo for the treatment of bladder tumors. Here, we evaluated the ability of bladder tumor cells to bind and endocytose FAP via fibronectin-integrin complexes. We found that microaggregation induced by an anti-FAP polyclonal antibody accelerated FAP uptake by T24 bladder tumor cells. FAP was determined to be internalized via a clathrin-independent, caveolae-dependent mechanism. Furthermore, once within the endosomal compartment, FAP was targeted to the lysosomal compartment with negligible recycling to the plasma membrane. Importantly, we demonstrated that FAP microaggregation and internalization could also be triggered by multivalent Ni(2+) NTA-bearing liposomes. Overall, our studies validate the use of FAP as a targeting vector and provide the foundation for the design of more effective, less-toxic bladder cancer therapeutics.

Lowe syndrome: Between primary cilia assembly and Rac1-mediated membrane remodeling.

Lowe syndrome (LS) is a lethal X-linked genetic disease caused by functional deficiencies of the phosphatidlyinositol 5-phosphatase, Ocrl1. In the past four years, our lab described the first Ocrl1-specific cellular phenotypes using dermal fibroblasts from LS patients. These phenotypes, validated in an ocrl1-morphant zebrafish model, included membrane remodeling (cell migration/spreading, fluid-phase uptake) defects and primary cilia assembly abnormalities. On one hand, our findings unraveled cellular phenotypes likely to be involved in the observed developmental defects; on the other hand, these discoveries established LS as a ciliopathy-associated disease. This article discusses the possible mechanisms by which loss of Ocrl1 function may affect RhoGTPase signaling pathways leading to actin cytoskeleton rearrangements that underlie the observed cellular phenotypes.

Epsins' novel role in cancer cell invasion.

The epsin family of endocytic adaptors has been found to be upregulated in cancer; however the relevance of these findings to this pathological condition is unclear. We have recently demonstrated that epsins are required for cell migration. In fact, epsin overexpression promotes cancer cell invasion. Further, and in agreement with our previous findings, we also observed that overexpression of epsins led to epithelial cell migration beyond colony boundaries. Additionally, our results show that epsin-3 is the most potent paralog enhancing cell migration and invasion. Interestingly, epsin-3 expression is not widespread but highly restricted to migratory keratinocytes and aggressive carcinomas. Upon further investigation, we also identified epsin-3 as being expressed in pancreatic cancer cells. These findings suggest that upregulation of the EPN3 gene is specifically associated with invasive, aggressive cancers. We predict that investigation of these links between the endocytic machinery and mechanisms involved in tumor dissemination will contribute to the development of novel anti-metastatic and anti-cancer strategies.

The epsin family of endocytic adaptors promotes fibrosarcoma migration and invasion.

Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient down-regulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral-binding protein 1). Epsin interacted with the N terminus of RalBP1 via its characteristic epsin N-terminal homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all the epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed up-regulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics.

Analysis of the development of a morphological phenotype as a function of protein concentration in budding yeast.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Lowe syndrome patient fibroblasts display Ocrl1-specific cell migration defects that cannot be rescued by the homologous Inpp5b phosphatase.

The Lowe syndrome (LS) is a life-threatening, developmental disease characterized by mental retardation, cataracts and renal failure. Although this human illness has been linked to defective function of the phosphatidylinositol 5-phosphatase, Ocrl1 (Oculo-Cerebro-Renal syndrome of Lowe protein 1), the mechanism by which this enzyme deficiency triggers the disease is not clear. Ocrl1 is known to localize mainly to the Golgi apparatus and endosomes, however it translocates to plasma membrane ruffles upon cell stimulation with growth factors. The functional implications of this inducible translocation to the plasma membrane are presently unknown. Here we show that Ocrl1 is required for proper cell migration, spreading and fluid-phase uptake in both established cell lines and human dermal fibroblasts. We found that primary fibroblasts from two patients diagnosed with LS displayed defects in these cellular processes. Importantly, these abnormalities were suppressed by expressing wild-type Ocrl1 but not by a phosphatase-deficient mutant. Interestingly, the homologous human PI-5-phosphatase, Inpp5b, was unable to complement the Ocrl1-dependent cell migration defect. Further, Ocrl1 variants that cannot bind the endocytic adaptor AP2 or clathrin, like Inpp5b, were less apt to rescue the migration phenotype. However, no defect in membrane recruitment of AP2/clathrin or in transferrin endocytosis by patient cells was detected. Collectively, our results suggest that Ocrl1, but not Inpp5b, is involved in ruffle-mediated membrane remodeling. Our results provide new elements for understanding how Ocrl1 deficiency leads to the abnormalities associated with the LS.

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway.

The epsins are a family of adaptors involved in recruiting other endocytic proteins, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. We have previously demonstrated that the ENTH domain of epsin is involved in Cdc42 signaling regulation. Here, we present evidence that yeast epsin 2 (Ent2) plays a signaling role during cell division. We observed that overexpression of the ENTH domain of Ent2 (ENTH2), but not Ent1, promoted the formation of chains of cells and aberrant septa. This dominant-negative effect resulted from ENTH2-mediated interference with septin assembly pathways. We mapped the ENTH2 determinants responsible for induction of the phenotype and found them to be important for efficient binding to the septin regulatory protein, Bem3. Supporting a physiological role for epsin 2 in cell division, the protein localized to sites of polarized growth and cytokinesis and rescued a defect in cell division induced by Bem3 misregulation. Collectively, our findings provide a potential molecular mechanism linking endocytosis (via epsin 2) with signaling pathways regulating cell division.