Mechanisms of Sorting and Trafficking for Melanosome Biogenesis as Revealed by Studies of the Human Pigment Cell Protein Oca2

Version:1.0 StartHTML:0000000224 EndHTML:0000004814 StartFragment:0000002412 EndFragment:0000004778 SourceURL:file://localhost/Users/hastha82/Desktop/temporary%20desktop%20folder/MARKS%20LAB/THESIS/final%20changes.doc Certain cell types harbor specialized lysosome-related organelles (LROs) that derive from the endocytic system like conventional lysosomes but have unique functions. The coexistence of LROs and lysosomes in some cell types implies the existence of sorting mechanisms that divert resident cargo proteins to LROs. Based on the pigment-synthesizing melanosome in melanocytes as a model LRO and two melanosomal resident proteins as model cargoes, current models suggest that cargoes are sorted from early endosomes to melanosomes via one of two independent pathways mediated by the multisubunit complexes AP-3 or BLOC-1, each of which is defective in subtypes of the LRO biogenesis disease Hermansky-Pudlak Syndrome (HPS). An AP-3-related protein complex, AP-1, is thought to function in concert with BLOC-1. In this thesis, I assess the pigment-cell-specific putative transporter protein OCA2, as a third potential cargo protein with which to further dissect the relationships between AP-3, AP-1 and BLOC-1 in melanosomal transport. I first investigate the localization and site of action of OCA2. I use biochemical approaches in combination with site-directed mutagenesis and indirect immunofluorescence microscopic analysis of exogenously-expressed OCA2 in melanocytes to show that OCA2 is indeed a melanosome resident protein and does not function within the endoplasmic reticulum as has been suggested by other models. I show that melanosome localization is essential for OCA2 function and requires an acidic dileucine motif in the N-terminal cytoplasmic domain that can bind to both AP-3 and AP-1. Using site-directed mutagenesis in combination with yeast three hybrid assays and immunofluorescence microscopy analyses in melanocytes derived from mouse models of HPS and controls, I define the features of the OCA2 sorting signal that direct binding to AP-1 or AP-3 and show that OCA2 requires both AP-3 interaction and BLOC-1 for melanosomal localization. My results resolve a controversy regarding OCA2 localization, shed light on the interplay between AP-1 and AP-3 in melanosomal trafficking, and provide the first direct evidence for cooperation between BLOC-1 and AP-3 in trafficking to a LRO

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 A and 2.0 A respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities

Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling

Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p–Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p–Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p–Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Background The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. Results The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. Conclusions Cry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities

Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system

A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins.

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not to other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack

Extracellular Signal-regulated Kinase Regulates Clathrin-independent Endosomal Trafficking

Extracellular signal-regulated kinase (Erk) is widely recognized for its central role in cell proliferation and motility. Although previous work has shown that Erk is localized at endosomal compartments, no role for Erk in regulating endosomal trafficking has been demonstrated. Here, we report that Erk signaling regulates trafficking through the clathrin-independent, ADP-ribosylation factor 6 (Arf6) GTPase-regulated endosomal pathway. Inactivation of Erk induced by a variety of methods leads to a dramatic expansion of the Arf6 endosomal recycling compartment, and intracellular accumulation of cargo, such as class I major histocompatibility complex, within the expanded endosome. Treatment of cells with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduces surface expression of MHCI without affecting its rate of endocytosis, suggesting that inactivation of Erk perturbs recycling. Furthermore, under conditions where Erk activity is inhibited, a large cohort of Erk, MEK, and the Erk scaffold kinase suppressor of Ras 1 accumulates at the Arf6 recycling compartment. The requirement for Erk was highly specific for this endocytic pathway, because its inhibition had no effect on trafficking of cargo of the classical clathrin-dependent pathway. These studies reveal a previously unappreciated link of Erk signaling to organelle dynamics and endosomal trafficking

AP2-mediated CD79-driven Endocytosis is Regulated in Cis via ITAM-embedded DCSM and QTAT motifs.

<p><u>Panel A</u>, Binding of AP2-btn to bead-captured GST bearing the indicated CD79a or CD79b cytoplasmic domains with the indicated mutations was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of wild type CD79a binding and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between the non-mutated CD79a or CD79b_DCSM cytoplasmic domains and other samples. <u>Panels B and C</u>, Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed and quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter protein expressing the wild type CD79a or CD79b cytoplasmic domains and other reporter proteins. <u>Panel D</u>, Diagrammatic representation of <i>cis</i> and <i>trans</i> regulation of BCR AP2 binding and endocytosis. The open arrows indicate <i>cis</i> regulation by the DCSM and QTAT regulatory motifs. The closed arrows represent <i>trans</i> regulation of the endocytic activity of each cytoplasmic domain by the presence of the partner cytoplasmic domain.</p

CD79b Y195 Controls BCR Internalization.

<p><u>Panel A</u>, Amino acid sequences of the cytoplasmic domains of CD79a and CD79b. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, Confocal laser scanning microscopic analysis of the endocytosis of indicated MHC class II-CD79 chimeric proteins. Plasma membrane is yellow (anti-MHC class II-Alexa 488+ post-endocytic labeling of external MHC class II-CD79 with an Alexa 594-labeled antibody). Internalized MHC class II-CD79 is green (anti-MHC class II-Alexa 488 only). <u>Panel C</u>, Quantification of MHC class II-CD79 endocytosis. 100+ cells from across 3 independent experiments were scored for internalization. Reported is the percent of cells showing internalized MHC class II-CD79 for each construct. Statistical comparisons were made between the construct with both CD79 cytoplasmic domains and cells expressing other constructs.</p

AP2µ Directly Binds the YxxØ Motif Centered on the Membrane-Proximal Tyrosine of Isolated CD79a.

<p><u>Panel A</u>, The binding of AP2µ-btn to bead-captured GST bearing 21 amino acid peptides centered on the five CD79 YxxØ motifs was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of CD79a–AP2 interactions and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between CD79a and other samples. <u>Panel B</u>, For both CD79a and CD79b, a positive or negative regulatory motif lies within +/−10 amino acids of the tyrosine residue of the membrane-proximal YxxØ AP2 binding motif. In this example, the motif is arbitrarily depicted as being downstream of the YxxØ motif.</p