Kathleen Boesze-Battaglia

A Novel Mode of Action for a Microbial-Derived Immunotoxin: The Cytolethal Distending Toxin Subunit B Exhibits Phosphatidylinositol 3,4,5-Triphosphate Phosphatase Activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G2 arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P3 to PI-3,4-P2 and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G2 arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P3 in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P3 levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P3. Finally, reduction of Jurkat cell PI-3,4,5-P3 synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P3 phosphatase activity, but also that toxicity in lymphocytes is related to this activity.

Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxin

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal‐distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt‐mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1‐enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl β‐cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed.

Association of a photoreceptor-specific tetraspanin protein, ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes.

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 °C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-β-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.

Cytolethal Distending Toxin-induced Cell Cycle Arrest of Lymphocytes Is Dependent upon Recognition and Binding to Cholesterol

Induction of cell cycle arrest in lymphocytes after exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. In this study we further demonstrate that the association of Cdt with lymphocyte plasma membranes is dependent upon binding to cholesterol. Depletion of cholesterol resulted in reduced toxin binding, whereas repletion of cholesterol-depleted cells restored binding. We employed fluorescence resonance energy transfer and surface plasmon resonance to demonstrate that toxin association with model membranes is dependent upon the concentration of cholesterol; moreover, these interactions were cholesterol-specific as the toxin failed to interact with model membranes containing stigmasterol, ergosterol, or lanosterol. Further analysis of the toxin indicated that the CdtC subunit contains a cholesterol recognition/interaction amino acid consensus (CRAC) region. Mutation of the CRAC site resulted in decreased binding of the holotoxin to cholesterol-containing model membranes as well as to the surface of Jurkat cells. The mutant toxin also exhibited reduced capacity for intracellular transfer of the active toxin subunit, CdtB, as well as reduced toxicity. Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for Cdt and that this association can be blocked by either depleting membranes of cholesterol or mutation of the CRAC site.

Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function‐associated antigen‐1 (LFA‐1)‐bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the β2 integrin, LFA‐1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA‐1‐deficient Jurkat mutant (Jβ2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+[Ca2+]c, an event that is independent of the Ltx/LFA‐1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA‐1 in cholesterol and sphingolipid‐rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA‐1 within lipid rafts is essential for cell lysis. Jβ2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin‐induced Ca2+ fluxes mobilize LFA‐1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.

Acidic Nanoparticles Are Trafficked to Lysosomes and Restore an Acidic Lysosomal pH and Degradative Function to Compromised ARPE-19 Cells

Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.

Photoreceptor renewal: a role for peripherin/rds.

Visual transduction begins with the detection of light within the photoreceptor cell layer of the retina. Within this layer, specialized cells, termed rods and cones, contain the proteins responsible for light capture and its transduction to nerve impulses. The phototransductive proteins reside within an outer segment region that is connected to an inner segment by a thin stalk rich in cytoskeletal elements. A unique property of the outer segments is the presence of an elaborate intracellular membrane system that holds the phototransduction proteins and provides the requisite lipid environment. The maintenance of normal physiological function requires that these postmitotic cells retain the unique structure of the outer segment regions--stacks of membrane saccules in the case of rods and a continuous infolding of membrane in the case of cones. Both photoreceptor rod and cone cells achieve this through a series of coordinated steps. As new membranous material is synthesized, transported, and incorporated into newly forming outer segment membranes, a compensatory shedding of older membranous material occurs, thereby maintaining the segment at a constant length. These processes are collectively referred to as ROS (rod outer segment) or COS (cone outer segment) renewal. We review the cellular and molecular events responsible for these renewal processes and present the recent but compelling evidence, drawn from molecular genetic, biochemical, and biophysical approaches, pointing to an essential role for a unique tetraspanning membrane protein, called peripherin/rds, in the processes of disk morphogenesis.

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.

Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: evidence for a role in retinal degenerations.

Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimers disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimers disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body.

Melanoregulin (MREG) modulates lysosome function in pigment epithelial cells.

Melanoregulin (MREG), the product of the Mregdsu gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg-/- mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg-/- mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking.