PEGylation of Carbonate Apatite Nanoparticles Prevents Opsonin Binding and Enhances Tumor Accumulation of Gemcitabine

pH sensitives carbonate apatite (CA) has emerged as a targeted delivery vehicle for chemotherapeutics agent with tremendous potential to increase the effectivity of breast cancer treatment. The major challenge for intravenous delivery of drug-incorporated nanoparticles is their rapid opsonization, resulting in accumulation within the organs of reticuloendothelial system, such as liver and spleen. Therefore, surface modification by polyethylene glycol was implemented to improve the half-life of drug-particle complexes and enhance their uptake by target tissues. A simple, rapid, and sensitive triple quadrupole liquid chromatography–mass spectrometry method was developed and validated for quantification of gemcitabine in plasma, various organs and tumor tissues of mice with breast carcinoma, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis, quadrupole-time of flight liquid chromatography–mass spectrometry and analysis by SwissProt.Mus_musculus database were performed for protein separation, identification, and homology search by comparing the de novo sequence tag. PEGylated CA exhibited almost 6-fold increase in gemcitabine accumulation in tumor with significant reduction in other organs within 1 h of intravenous administration, compared to free drug. In addition, plasma drug amount was found to be higher in PEGylated particles, implying their role in prolonging blood circulation time of particle-bound gemcitabine. Investigation of protein corona composition demonstrated notable reduction in opsonin interactions after PEGylation of CA particles. Overall, the results indicate that the composition and dynamics of protein corona subjected to alteration by PEGylation play crucial roles in affecting successful nanoparticle-based targeted delivery of a cytotoxic drug.     

2D-DIGE proteomic analysis identifies new potential therapeutic targets for adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare aggressive tumor with poor prognosis when metastatic at diagnosis. The tumor biology is still mostly unclear, justifying the limited specificity and efficacy of the anti-cancer drugs currently available. This study reports the first proteomic analysis of ACC by using two-dimensional-differential-in-gel-electrophoresis (2D-DIGE) to evaluate a differential protein expression profile between adrenocortical carcinoma and normal adrenal. Mass spectrometry, associated with 2D-DIGE analysis of carcinomas and normal adrenals, identified 22 proteins in 27 differentially expressed 2D spots, mostly overexpressed in ACC. Gene ontology analysis revealed that most of the proteins concurs towards a metabolic shift, called the Warburg effect, in adrenocortical cancer. The differential expression was validated by Western blot for Aldehyde-dehydrogenase-6-A1,Transferrin, Fascin-1,Lamin A/C,Adenylate-cyclase-associated-protein-1 and Ferredoxin-reductase. Moreover, immunohistochemistry performed on paraffin-embedded ACC and normal adrenal specimens confirmed marked positive staining for all 6 proteins diffusely expressed by neoplastic cells, compared with normal adrenal cortex.In conclusion, our preliminary findings reveal a different proteomic profile in adrenocortical carcinoma compared with normal adrenal cortex characterized by overexpression of mainly metabolic enzymes, thus suggesting the Warburg effect also occurs in ACC. These proteins may represent promising novel ACC biomarkers and potential therapeutic targets if validated in larger cohorts of patients.     

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro,in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.     

Data-independent acquisition-based quantitative proteomic analysis of m.3243A>G MELAS reveals novel potential pathogenesis and therapeutic targets

The pathogenesis of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS) syndrome has not been fully elucidated. The m.3243A > G mutation which is responsible for 80% MELAS patients affects proteins with undetermined functions. Therefore, we performed quantitative proteomic analysis on skeletal muscle specimens from MELAS patients. We recruited 10 patients with definitive MELAS and 10 age- and gender- matched controls. Proteomic analysis based on nanospray liquid chromatography-mass spectrometry (LC-MS) was performed using data-independent acquisition (DIA) method and differentially expressed proteins were revealed by bioinformatics analysis. We identified 128 differential proteins between MELAS and controls, including 68 down-regulated proteins and 60 up-regulated proteins. The differential proteins involved in oxidative stress were identified, including heat shock protein beta-1 (HSPB1), alpha-crystallin B chain (CRYAB), heme oxygenase 1 (HMOX1), glucose-6-phosphate dehydrogenase (G6PD) and selenoprotein P. Gene ontology and kyoto encyclopedia of genes and genomes pathway analysis showed significant enrichment in phagosome, ribosome and peroxisome proliferator-activated receptors (PPAR) signaling pathway. The imbalance between oxidative stress and antioxidant defense, the activation of autophagosomes, and the abnormal metabolism of mitochondrial ribosome proteins (MRPs) might play an important role in m.3243A > G MELAS. The combination of proteomic and bioinformatics analysis could contribute potential molecular networks to the pathogenesis of MELAS in a comprehensive manner.     

Proximity proteomics of synaptopodin provides insight into the molecular composition of the spine apparatus of dendritic spines

The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus–specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.

The neuronal endoplasmic reticulum (ER) is an intricate continuous network of membrane tubules and cisterns that runs throughout neuronal processes with region-specific specializations. One such specialization of the smooth ER is the spine apparatus (SA) that is located in a subset of dendritic spines. The SA consists of stacks of flat cisterns that are connected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft (1–3) (Fig. 1A and SI Appendix, Fig. S1A). Morphological changes in the SA have been reported after long-term potentiation (4) and also, in a variety of human disorders, including several neurodegenerative conditions (5–9). While the first observation of the SA by electron microscopy (EM) was reported in 1959 by Gray (1), our understanding of this organelle remains fairly limited. Its molecular characterization has proven to be challenging due to the difficulty of its biochemical isolation and its absence in organisms suitable for genetic screens.Open in a separate windowFig. 1.The localization of synaptopodin (indicated as Synpo in all figures) in cultured hippocampal neurons overlaps with the localization of the SA in dendritic spines of cortical slices. (A) SA as visualized by transmission EM. (B and C) SA and ER reconstructed by a semiautomated algorithm from 3D volumes acquired by FIB-SEM. An SA is shown in B, while C shows a portion of a dendritic shaft with spines containing (magenta arrows) and not containing (white arrows) an SA. The plasma membrane (PM) is shown in blue, the ER is in red, and the post-synaptic density (PSD) is in yellow. (D) Cisternal organelle (CO), as observed in an FIB-SEM optical section, at an axonal initial segment. Note that the stacks of ER cisterns are similar to those characteristics of the SA. (E) mRFP-synaptopodin coexpressed with cytosolic EGFP as a marker of the entire dendritic volume. Note in the zoomed-in views of the region enclosed by a rectangle (the lower three fields) that synaptopodin is concentrated near the spine neck, where the SA is localized. (F) Localization by immunofluorescence of endogenous synaptopodin showing strong overlap with a pool of F-actin labeled by phalloidin-Alexa488. Also, in this sample, the magnified views (the lower three fields) show enrichment of synaptopodin, relative to actin, at the spine neck. (G) mRFP-synaptopodin coexpressed with an ER marker, EGFP-VAPB, showing colocalization of the two proteins. In the zoomed-in views (the lower three fields), red arrows show a spine positive for both the ER marker and synaptopodin, and the blue arrows show a spine with the ER marker but lacking synaptopodin. (H) Percentage of ER-positive spines that also contain synaptopodin and percentage of synaptopodin-positive spines that contain ER quantified in cultured hippocampal neurons expressing mRFP-synaptopodin and the ER markers EGFP-VAPB or EGFP-Sec61β. Each data point represents at least 99 spines from a single neuron. (I) Spine of a synaptopodin KO mouse that lacks the SA but contains the ER. (J) Quantification of the number of spines where ER was visible in the plane of the section with or without an SA in the brain of wild type (WT) vs. synaptopodin mutant mice (n≥800 spines per genotype).The only known protein enriched at the SA and required for its formation is synaptopodin, a protein without transmembrane regions localized in the cytosolic space (10). Neuronal synaptopodin specifically localizes to dendritic spines and to the axonal initial segment, where another specialization of the ER similar to the SA (stack of flattened cisterns) called the cisternal organelle is present (11–14). Lack of synaptopodin in synaptopodin knock-out (KO) mice correlates with the lack of SA and of the cisternal organelle, as well as with a reduction in Hebbian plasticity and spatial memory (11, 15–18). A longer isoform of synaptopodin is expressed in the foot processes of podocytes, where it functions as a regulator of the actin cytoskeleton (19, 20). Synaptopodin binds to and bundles actin (21) and interacts with several actin binding proteins, such as α-actinin (13, 21, 22). While more is known about the interactors of synaptopodin in podocytes, its binding partners at the SA remain unknown.The goal of this work was to gain insight into the molecular composition of the SA. To this aim, we used synaptopodin as a starting point for our analysis. We identified some of its binding partners by an in vivo proximity biotinylation approach and characterized the specific localization of a subset of these proteins in neurons and their interaction with synaptopodin in an exogenous system.