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Phosphorylation of α-synuclein at the Serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of total α-syn is phosphorylated at this site, and consequently, almost all studies to date have focused on putative pathologic roles of this post-translational modification. We noticed that unlike native (total) α-syn that is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of α-syn to attenuate activity-dependent synaptic vesicle (SV) recycling – widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments α-syn Ser-129P, and this phosphorylation is required for α-syn binding to VAMP2 and synapsin – two functional binding-partners that are necessary for α-syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilizes this region and facilitates protein-protein interactions. Our experiments indicate that the pathology-associated Ser129P is an unexpected physiologic trigger of α-syn function, which has broad implications for pathophysiology and drug-development.

Epsins are endocytic adaptor proteins involved in the internalization of important membrane proteins such as EGFR and Notch ligands. Therefore, this protein family impacts critical signaling pathways and processes such as cell migration and cytokinesis and is ultimately required for embryo development in mammals and cell viability in yeast. Intriguingly, although Epsins are conserved and display similar binding determinants, the process of endocytosis in yeast and mammals exhibit some dramatic mechanistic differences. Therefore, we wondered if the function of Epsins in these organisms are similar and are similarly regulated or they also differ. Since proper and timely localization is needed for function, we determined what elements target Epsins to endocytic sites in yeast vs mammals. Specifically, using a systematic/combinatorial mutagenesis approach we produced a collection of yeast and human Epsin mutated variants that was tested for localization at endocytic sites and for function.Our results showed that the intrinsically disordered carboxy-terminus holds the major determinants (involved in binding of ubiquitin, AP2, clathrin and EH domain-containing proteins) for proper intracellular localization of different Epsin paralogs and homologs in yeast and mammals, while also having a major impact on function. Importantly, we established hierarchies of carboxy-terminal binding determinants for sustaining Epsin localization which turned to be different for human vs. yeast cells; favoring clathrin and AP2 binding in the former and recognition of cargo and EH domain-containing proteins for the latter. Further, we found evidence in both systems that yeast Epsins also use for localization regions of the protein that were until now of unknown functional relevance, i.e., glutamine-rich sequences. Interestingly, some molecular determinants within the Epsin molecule seem to have functional importance beyond its contribution to localization to endocytic sites. Based on these findings, we propose working models for Epsin function and recruitment to membranes/endocytic sites at different maturation stages.

Lowe Syndrome (LS) is a lethal genetic disorder caused by mutations in theOCRL1gene which encodes the lipid 5’ phosphatase Ocrl1. Patients exhibit a characteristic triad of symptoms including eyes, brain and kidneys abnormalities with renal failure as the most common cause of premature death. Over 200OCRL1mutations 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.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 can help stratify patients as well as to produce a more accurate prognosis depending on the nature and location of the mutation within theOCRL1gene.

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.

Epsins are endocytic adaptor proteins with signaling and endocytic functions. The three mammalian epsin paralogs are made of an Epsin N-Terminal Homology (ENTH) domain and an unstructured C-terminal region. The highly conserved ENTH domain plays a role in signaling by blocking RhoGAP activity and is required for cell migration in mammalian cells. However, our lab has previously shown that only epsin full length overexpression can enhance cell migration, but the ENTH domain alone cannot. Among the three Epsin paralogs, epsin 3 followed by epsin 2 were able to substantially enhance cell migration. This study is the first one to systematically and comprehensibly address the contribution of different motifs within the epsin C-terminus to enhance protein localization and cell migration. We show that is not the lipid-binding ENTH domain, but the C-terminus of epsin the one playing a major role in epsin association with sites of endocytosis. Further, we dissected the contribution of individual C-terminal endocytic (clathrin-, AP2-, Ubiquitin- and EH domain-binding) motifs for epsin localization. We found that while all motifs show a degree of synergism, the clathrin-binding motifs are the most important for epsin localization. Our study also showed that, these motifs (particularly the clathrin binding site) play an important role in sustaining endocytic site dynamics and cell migration.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC(50) < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

Cell-autonomous and cell-nonautonomous mechanisms of neurodegeneration appear to occur in the proteinopathies, including Alzheimer’s and Parkinson’s diseases. However, how neuronal toxicity is generated from misfolding-prone proteins secreted by nonneuronal tissues and whether modulating protein aggregate levels at distal locales affects the degeneration of postmitotic neurons remains unknown. We generated and characterized animal models of the transthyretin (TTR) amyloidoses that faithfully recapitulate cell-nonautonomous neuronal proteotoxicity by expressing human TTR in the Caenorhabditis elegans muscle. We identified sensory neurons with affected morphological and behavioral nociception-sensing impairments. Nonnative TTR oligomer load and neurotoxicity increased following inhibition of TTR degradation in distal macrophage-like nonaffected cells. Moreover, reducing TTR levels by RNAi or by kinetically stabilizing natively folded TTR pharmacologically decreased TTR aggregate load and attenuated neuronal dysfunction. These findings reveal a critical role for in trans modulation of aggregation-prone degradation that directly affects postmitotic tissue degeneration observed in the proteinopathies.

The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle-dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multiorgan diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies appears to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e. affecting cilia assembly) will lead to more severe symptoms, whereas those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology.

The complex biochemical activities that take place within the cell require a precise spatiotemporal organization of its components. This dynamic intracellular order is generated and maintained by a transport system based on membrane-bound carriers that pinch off from a donor and deliver cargoes into a target compartment. The crucial task of selecting the cargoes to be transported from one compartment to another is the responsibility of the protein machinery that coats the cytosolic side of the carriers until they bud off. These coats contain specialized components known as ‘adaptors’ that interpret cargo destination information and include them or not into the nascent transport carrier. This article reviews several families of adaptors and summarizes their function, specificity, and cellular/organism relevance.

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.

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.

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.

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.

The primary cilium (PC) is a plasma membrane-derived structure of great importance for cell and organismal physiology. Indeed, abnormalities in assembly or function of the PC trigger the onset of a group of genetic diseases collectively known as ciliopathies. In recent years, it has become evident that the integrity and function of the PC depends substantially on signaling elements such as phosphoinositides (PI) and their regulators. Because phospholipids such as PI(4,5)P 2 constitute recruitment platforms for cytoskeleton, signaling, and trafficking machinery, control over their levels is critical for PC function. Although information about phosphoinositol phosphate (PIP) kinases in the PC is scarce, a growing body of evidence sup- ports a role for PIP phosphatases in cilia assembly/maintenance. Indeed, deficiencies in two 5' PIP phosphatases, Inpp5E and Ocrl1, are clearly linked to ciliopathies like Joubert/MORM syndromes, or ciliopathy-associated dise ases like Lowe syndrome. Here, we review the unique roles of these proteins and their specific site of action for ensuring ciliary integrity. Further, we discuss the possibility that a phosphatase relay system able to pass PI control from a preciliary

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.

One of the most common links between cancer and endocytosis is defective internalization of Receptor Tyrosine Kinases, for example, linked to a deficit in endocytic proteins. This scenario leads to hypersignaling and in turn to malignant transformation. Nevertheless, there are also cases in which endocytic proteins, such as the epsin adaptors, are found upregulated in invasive cancers. Indeed, we demonstrated that overexpression of the epsins enhances cancer cell migration and invasion. Furthermore, depletion of epsins (by siRNA) led to impairment of cancer cell invasion. However, the different epsin paralogs varied in their capacity to enhance migration/invasion in the following order: epsin 3 > epsin2 >> epsin1. The epsin paralogs have a highly conserved Epsin N-terminal Homology or the ENTH domain (N-terminus) and a C-terminus where the most divergent regions are housed. While our data shows that both the ENTH domain and the C-terminus are required for enhancing migration, we speculate that the differential ability of the paralogs to migrate/invade lies in their less conserved C-terminus region. This study aims to uncover the mechanism by which epsin overexpression leads to enhanced migration/invasion and to identify the regions of the epsin C-terminus which gives the paralogs their differential ability to enhance migration/invasion. Here we describe a systematic combinatorial approach for mapping the epsin determinants involved in localizing epsin at endocytic sites and required for its effect on cancer cell migration/invasion. Our data clearly shows that Clathrin binding motifs (out of the 4 types of endocytic determinants) are the strongest localization determinants of epsin2, however are not sufficient. Infact, 2 types of endocytic determinants are required for proper epsin2 localization suggesting co-operativity and redundancy. We also found that mutation of any 2 types of endocytic determinants can also reduce the ability of epsin2 to migrate as compared to the wild-type protein. Expanding the results obtained from epsin2 to epsin3 will reveal the similarities and differences between the paralogs. Taking together our findings and the requirement of these proteins for cancer cell invasion, we envision epsin2 and epsin3 as targets for novel anti-cancer and anti-metastatic approaches. Citation Format: Kayalvizhi Madhivanan, R. Claudio Aguilar. Role of the endocytic adaptor Epsin in cancer cell migration. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A46.