Andrea Bugarcic

The Vps35 D620N mutation linked to Parkinson's disease disrupts the cargo sorting function of retromer

The retromer is a trimeric cargo‐recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinsons disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromers subcellular localization and its capacity to sort cargo. We show that expression of the PD‐linked Vps35 D620N mutant redistributes retromer‐positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild‐type Vps35. While PD‐linked point mutant Vps35 D620N interacts with the cation‐independent mannose‐6‐phosphate receptor (CI‐M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI‐M6PR ligand and protease responsible for degradation of α‐synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.

Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans

Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.

Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases

Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr–sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.

Rotavirus Nonstructural Glycoprotein NSP4 Is Secreted from the Apical Surfaces of Polarized Epithelial Cells

ABSTRACT NSP4, a nonstructural glycoprotein encoded by rotavirus, is involved in the morphogenesis of virus particles in the endoplasmic reticulum of infected cells. NSP4 is also implicated in the pathophysiology of rotavirus-induced diarrhea by acting as an enterotoxin. To mediate enterotoxic effects in vivo, NSP4 must be secreted or released from rotavirus-infected cells in a soluble form; however, previous studies have indicated that NSP4 is a transmembrane glycoprotein localized within endomembrane compartments in infected cells. In this study, we examined the fate of NSP4 synthesized in Caco-2 cells infected with bovine rotavirus. Our studies reveal that NSP4 is actively secreted into the culture medium, preferentially from the infected-cell apical surface. The secretion of NSP4 is dramatically inhibited by brefeldin A and monensin, suggesting that a Golgi-dependent pathway is involved in release of the protein. In agreement with the proposed involvement of the Golgi apparatus during secretion, secreted NSP4 appears to undergo additional posttranslational modification compared to its cell-associated counterpart and is partially resistant to deglycosylation by endoglycosidase H. Our experiments identify a novel, soluble form of NSP4 secreted from virus-infected cells with the potential to carry out the enterotoxigenic role previously attributed to recombinant forms of the protein.

Vps26A and Vps26B Subunits Define Distinct Retromer Complexes

The trimeric Vps29–Vps35–Vps26 sub‐complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans‐Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B‐retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A‐myc or Vps26B‐myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian‐specific Vps26 paralogues. Vps26B‐retromer, like Vps26A‐retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B‐retromer and cation‐independent mannose 6‐phosphate receptor (CI‐M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B‐retromer with maturing endosomes relative to Vps26A‐retromer. Interestingly, the cycling of CI‐M6PR is restored upon deletion of the variable Vps26B C‐terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C‐terminal region can control cargo selection of the Vps26B‐retromer.

Parkinson disease-linked Vps35 R524W mutation impairs the endosomal association of retromer and induces α-synuclein aggregation

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.

Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein.

The rotavirus spike protein VP4 mediates attachment to host cells and subsequent membrane penetration. The VP8(*) domain of VP4 forms the spike tips and is proposed to recognize host-cell surface glycans. For sialidase-sensitive rotaviruses such as rhesus (RRV), this recognition involves terminal sialic acids. We show here that the RRV VP8(*)(64-224) protein competes with RRV infection of host cells, demonstrating its relevance to infection. In addition, we observe that the amino acids revealed by X-ray crystallography to be in direct contact with the bound sialic acid derivative methyl alpha-D-N-acetylneuraminide, and that are highly conserved amongst sialidase-sensitive rotaviruses, are residues that are also important in interactions with host-cell carbohydrates. Residues Arg101 and Ser190 of the RRV VP8(*) carbohydrate-binding site were mutated to assess their importance for binding to the sialic acid derivative and their competition with RRV infection of host cells. The crystallographic structure of the Arg(101)Ala mutant crystallized in the presence of the sialic acid derivative was determined at 295 K to a resolution of 1.9 A. Our multidisciplinary study using X-ray crystallography, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and competitive virus infectivity assays to investigate RRV wild-type and mutant VP8(*) proteins has provided the first evidence that the carbohydrate-binding cavity in RRV VP8(*) is used for host-cell recognition, and this interaction is not only with the sialic acid portion but also with other parts of the glycan structure.

A Set of Vertically Integrated Inquiry-Based Practical Curricula that Develop Scientific Thinking Skills for Large Cohorts of Undergraduate Students.

Science graduates require critical thinking skills to deal with the complex problems they will face in their 21st century workplaces. Inquiry-based curricula can provide students with the opportunities to develop such critical thinking skills; however, evidence suggests that an inappropriate level of autonomy provided to underprepared students may not only be daunting to students but also detrimental to their learning. After a major review of the Bachelor of Science, we developed, implemented, and evaluated a series of three vertically integrated courses with inquiry-style laboratory practicals for early-stage undergraduate students in biomedical science. These practical curricula were designed so that students would work with increasing autonomy and ownership of their research projects to develop increasingly advanced scientific thinking and communication skills. Students undertaking the first iteration of these three vertically integrated courses reported learning gains in course content as well as skills in scientific writing, hypothesis construction, experimental design, data analysis, and interpreting results. Students also demonstrated increasing skills in both hypothesis formulation and communication of findings as a result of participating in the inquiry-based curricula and completing the associated practical assessment tasks. Here, we report the specific aspects of the curricula that students reported as having the greatest impact on their learning and the particular elements of hypothesis formulation and communication of findings that were more challenging for students to master. These findings provide important implications for science educators concerned with designing curricula to promote scientific thinking and communication skills alongside content acquisition.

An inquiry-based practical for a large, foundation-level undergraduate laboratory that enhances student understanding of basic cellular concepts and scientific experimental design

Student‐centered education involving research experiences or inquiry have been shown to help undergraduate students understand, and become excited about, the process of scientific investigation. These benefits are particularly important for students in the early stages of their degree (Report and Kenny, http://naplesccsunysbedu/Pres/boyernsf/1998). However, embedding such experiences into the curriculum is particularly difficult when dealing with early stage students, who are in larger cohorts and often lack the background content knowledge necessary to engage with primary research literature and research level methods and equipment. We report here the design, delivery, assessment, and subsequent student learning outcomes of a 4‐week practical module for 120 students at the beginning of their second year of university, which successfully engages students in designing cell culture experiments and in understanding the molecular processes and machinery involved in the basic cellular process of macropinocytosis.