Sara Sheibani

Crucial role of the protein corona for the specific targeting of nanoparticles

AIMS We aimed to investigate the physicochemical effects of superparamagnetic iron oxide nanoparticles (SPIONs) on the composition of the protein corona and their correspondence toxicological issues. MATERIALS & METHODS SPIONs of different sizes and surface charges were exposed to fetal bovine serum. The structure/composition and biological effects of the protein corona-SPION complexes were probed. RESULTS & DISCUSSION The affinity and level of adsorption of specific proteins is strongly dependent on the size and surface charge of the SPIONs. In vivo experiments on the mouse blood-brain barrier model revealed that nontargeted SPIONs containing specific proteins will enter the brain endothelial barrier cells. CONCLUSION Some commercially available nanoparticles used for target-specific applications may have unintended uptake in the body (e.g., brain tissue) with potential cytotoxity.

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death’. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.

Macromitophagy, neutral lipids synthesis, and peroxisomal fatty acid oxidation protect yeast from "liponecrosis", a previously unknown form of programmed cell death.

We identified a form of cell death called “liponecrosis.” It can be elicited by an exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA). Our data imply that liponecrosis is: (1) a programmed, regulated form of cell death rather than an accidental, unregulated cellular process and (2) an age-related form of cell death. Cells committed to liponecrotic death: (1) do not exhibit features characteristic of apoptotic cell death; (2) do not display plasma membrane rupture, a hallmark of programmed necrotic cell death; (3) akin to cells committed to necrotic cell death, exhibit an increased permeability of the plasma membrane for propidium iodide; (4) do not display excessive cytoplasmic vacuolization, a hallmark of autophagic cell death; (5) akin to cells committed to autophagic death, exhibit a non-selective en masse degradation of cellular organelles and require the cytosolic serine/threonine protein kinase Atg1p for executing the death program; and (6) display a hallmark feature that has not been reported for any of the currently known cell death modalities—namely, an excessive accumulation of lipid droplets where non-esterified fatty acids (including POA) are deposited in the form of neutral lipids. We therefore concluded that liponecrotic cell death subroutine differs from the currently known subroutines of programmed cell death. Our data suggest a hypothesis that liponecrosis is a cell death module dynamically integrated into a so-called programmed cell death network, which also includes the apoptotic, necrotic, and autophagic modules of programmed cell death. Based on our findings, we propose a mechanism underlying liponecrosis.

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.

Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells

The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H(2)O(2) and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H(2)O(2) in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.

Investigation of the Viability, Adhesion, and Migration of Human Fibroblasts in a Hyaluronic Acid/Gelatin Microgel-Reinforced Composite Hydrogel for Vocal Fold Tissue Regeneration.

The potential use of a novel scaffold biomaterial consisting of cross-linked hyaluronic acid (HA)-gelatin (Ge) composite microgels is investigated for use in treating vocal fold injury and scarring. Cell adhesion integrins and kinematics of cell motion are investigated in 2D and 3D culture conditions, respectively. Human vocal fold fibroblast (hVFF) cells are seeded on HA-Ge microgels attached to a HA hydrogel thin film. The results show that hVFF cells establish effective adhesion to HA-Ge microgels through the ubiquitous expression of β1 integrin in the cell membrane. The microgels are then encapsulated in a 3D HA hydrogel for the study of cell migration. The cells within the HA-Ge microgel-reinforced composite hydrogel (MRCH) scaffold have an average motility speed of 0.24 ± 0.08 μm min(-1) . The recorded microscopic images reveal features that are presumably associated with lobopodial and lamellipodial cell migration modes within the MRCH scaffold. Average cell speed during lobopodial migration is greater than that during lamellipodial migration. The cells move faster in the MRCH than in the HA-Ge gel without microgels. These findings support the hypothesis that HA-Ge MRCH promotes cell adhesion and migration; thereby they constitute a promising biomaterial for vocal fold repair.

Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

Sensing of Alzheimer's Disease and Multiple Sclerosis Using Nano-Bio Interfaces.

It is well understood that patients with different diseases may have a variety of specific proteins (e.g., type, amount, and configuration) in their plasmas. When nanoparticles (NPs) are exposed to these plasmas, the resulting coronas may incorporate some of the disease-specific proteins. Using gold (Au) NPs with different surface properties and corona composition, we have developed a technology for the discrimination and detection of two neurodegenerative diseases, Alzheimers disease (AD) and multiple sclerosis (MS). Applying a variety of techniques, including UV-visible spectra, colorimetric response analyses and liquid chromatography-tandem mass spectrometry, we found the corona-NP complexes, obtained from different human serums, had distinct protein composition, including some specific proteins that are known as AD and MS biomarkers. The colorimetric responses, analyzed by chemometrics and statistical methods, demonstrate promising capabilities of the technology to unambiguously identify and discriminate AD and MS. The developed colorimetric technology might enable a simple, inexpensive and rapid detection/discrimination of neurodegenerative diseases.

Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast

We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.