Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Nodular lymphocyte predominant Hodgkin lymphoma: Experience of Polish Pediatric Leukemia/Lymphoma Study Group.

Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) is a rare clinical entity. To investigate NLPHL clinical course and treatment a survey was performed within Polish Pediatric Leukaemia/Lymphoma Study Group (PPLLSG) participating centers. A questionnaire was sent to all participating centers and analysis of clinical data was performed. From 2010 to 2019, 19 pediatric patients with confirmed NLPHL were registered in Poland. Median age of patients was 12.2 (5.5 - 17.8) years. NLPHL occurred mainly in males (n = 17). Most of the patients (n = 16) had early stage disease - Stage I (n = 6) and stage II (n = 10). Four of the six patients with stage I disease (I A, n = 5; I B, n = 1) underwent complete primary resection. One of these relapsed and was treated with CVP (cyclophosphamide, vinblastine, prednisone) chemotherapy. Two other patients who were not resected completely received CVP chemotherapy and no relapses were observed. Thirteen patients presented with unresectable disease. Of these, eight received three CVP chemotherapy cycles, and five were treated with other chemotherapy regimens. Three relapses were observed and these patients were further treated with chemotherapy and rituximab. One patient underwent autologous stem cell transplantation (auto-SCT). All patients remain alive. Five-year progression-free survival and overall survival for the entire group of patients was 81.6% and 100%, respectively. NLPHL treatment results are consistent with results noted in other countries. Early stage patients have very good outcomes with surgery and observation or low intensity chemotherapy, but this approach may be insufficient in advanced disease.

The Regulation of Collagen Processing by miRNAs in Disease and Possible Implications for Bone Turnover.

This article describes several recent examples of miRNA governing the regulation of the gene expression involved in bone matrix construction. We present the impact of miRNA on the subsequent steps in the formation of collagen type I. Collagen type I is a main factor of mechanical bone stiffness because it constitutes 90-95% of the organic components of the bone. Therefore, the precise epigenetic regulation of collagen formation may have a significant influence on bone structure. We also describe miRNA involvement in the expression of genes, the protein products of which participate in collagen maturation in various tissues and cancer cells. We show how non-collagenous proteins in the extracellular matrix are epigenetically regulated by miRNA in bone and other tissues. We also delineate collagen mineralisation in bones by factors that depend on miRNA molecules. This review reveals the tissue variability of miRNA regulation at different levels of collagen maturation and mineralisation. The functionality of collagen mRNA regulation by miRNA, as proven in other tissues, has not yet been shown in osteoblasts. Several collagen-regulating miRNAs are co-expressed with collagen in bone. We suggest that collagen mRNA regulation by miRNA could also be potentially important in bone metabolism.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

Elucidating Key Motifs Required for Arp2/3-Dependent and Independent Actin Nucleation by Las17/WASP.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. Arp2/3 is a well-characterised protein complex that can promote nucleation of new filaments, though its activity requires additional nucleation promotion factors (NPFs). The best recognized of these factors are the WASP family of proteins that contain binding motifs for both monomeric actin and for Arp2/3. Previously we demonstrated that the yeast WASP homologue, Las17, in addition to activating Arp2/3 can also nucleate actin filaments de novo, independently of Arp2/3. This activity is dependent on its polyproline rich region. Through biochemical and in vivo analysis we have now identified key motifs within the polyproline region that are required for nucleation and elongation of actin filaments, and have addressed the role of the WH2 domain in the context of actin nucleation without Arp2/3. We have also demonstrated that full length Las17 is able to bind liposomes giving rise to the possibility of direct linkage of nascent actin filaments to specific membrane sites to which Las17 has been recruited. Overall, we propose that Las17 functions as the key initiator of de novo actin filament formation at endocytic sites by nucleating, elongating and tethering nascent filaments which then serve as a platform for Arp2/3 recruitment and function.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress.

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.

Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis.

Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins.

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology.

A novel actin-binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3.

BACKGROUND: Actin nucleation is the key rate-limiting step in actin polymerization, and tight regulation of this process is critical to ensure that actin filaments form only at specific regions of the cell. Las17 is the primary activator of Arp2/3-driven actin nucleation in yeast and is required for membrane invagination during endocytosis. Its mammalian homolog, WASP, has also been studied extensively as an activator of Arp2/3-driven actin polymerization. In both Las17 and WASP, actin nucleation activity is attributed to an ability to bind actin through a WH2 domain and to bind Arp2/3 through an acidic region. The central region of both Las17 and WASP is rich in proline residues and is generally considered to bind to SH3-domain-containing proteins. RESULTS: We have identified a novel actin-binding activity in the polyproline domain of both yeast Las17 and mammalian WASP. The polyproline domain of Las17 is also able to nucleate actin filaments independently of Arp2/3. Mutational analysis reveals that proline residues are required for this nucleation activity and that the binding site on actin maps to a region distinct from those used by other nucleation activities. In vivo analysis of yeast strains expressing las17 mutated in the WH2 domain, one of its proline motifs, or both shows additive defects in actin organization and endocytosis, with the proline mutant conferring more severe phenotypes than the WH2 mutant. CONCLUSIONS: Our data demonstrate a new actin-binding and nucleation mechanism in Las17/WASP that is required for its function in actin regulation during endocytosis.