Tunneling nanotube-mediated intercellular vesicle and protein transfer in the stroma-provided imatinib resistance in chronic myeloid leukemia cells

Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor–ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)—long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk—are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells

Global analysis of S-nitrosylation sites in the wild type and APP transgenic mouse brain-clues for synaptic pathology

Alzheimer’s disease (AD) is characterized by an early synaptic loss, which strongly correlates with the severity of dementia. The pathogenesis and causes of characteristic AD symptoms are not fully understood. Defects in various cellular cascades were suggested, including the imbalance in production of reactive oxygen and nitrogen species. Alterations in S-nitrosylation of several proteins were previously demonstrated in various AD animal models and patients. In this work, using combined biotinswitch affinity/nano-LC-MS/MS and bioinformatic approaches we profiled endogenous S-nitrosylation of brain synaptosomal proteins from wild type and transgenic mice overexpressing mutated human Amyloid Precursor Protein (hAPP). Our data suggest involvement of S-nitrosylation in the regulation of 138 synaptic proteins, including MAGUK, CamkII, or synaptotagmins. Thirty-eight proteins were differentially S-nitrosylated in hAPP mice only. Ninety-five S-nitrosylated peptides were identified for the first time (40% of total, including 33 peptides exclusively in hAPP synaptosomes). We verified differential S-nitrosylation of 10 (26% of all identified) synaptosomal proteins from hAPP mice, by Western blotting with specific antibodies. Functional enrichment analysis linked S-nitrosylated proteins to various cellular pathways, including: glycolysis, gluconeogenesis, calcium homeostasis, ion, and vesicle transport, suggesting a basic role of this post-translational modification in the regulation of synapses. The linkage of SNO-proteins to axonal guidance and other processes related to APP metabolism exclusively in the hAPP brain, implicates S-nitrosylation in the pathogenesis of Alzheimer’s disease.

Intracellular Protein S-Nitrosylation—A Cells Response to Extracellular S100B and RAGE Receptor

Human S100B is a small, multifunctional protein. Its activity, inside and outside cells, contributes to the biology of the brain, muscle, skin, and adipocyte tissues. Overexpression of S100B occurs in Down Syndrome, Alzheimer’s disease, Creutzfeldt–Jakob disease, schizophrenia, multiple sclerosis, brain tumors, epilepsy, melanoma, myocardial infarction, muscle disorders, and sarcopenia. Modulating the activities of S100B, related to human diseases, without disturbing its physiological functions, is vital for drug and therapy design. This work focuses on the extracellular activity of S100B and one of its receptors, the Receptor for Advanced Glycation End products (RAGE). The functional outcome of extracellular S100B, partially, depends on the activation of intracellular signaling pathways. Here, we used Biotin Switch Technique enrichment and mass-spectrometry-based proteomics to show that the appearance of the S100B protein in the extracellular milieu of the mammalian Chinese Hamster Ovary (CHO) cells, and expression of the membrane-bound RAGE receptor, lead to changes in the intracellular S-nitrosylation of, at least, more than a hundred proteins. Treatment of the wild-type CHO cells with nanomolar or micromolar concentrations of extracellular S100B modulates the sets of S-nitrosylation targets inside cells. The cellular S-nitrosome is tuned differently, depending on the presence or absence of stable RAGE receptor expression. The presented results are a proof-of-concept study, suggesting that S-nitrosylation, like other post-translational modifications, should be considered in future research, and in developing tailored therapies for S100B and RAGE receptor-related diseases

Insights Into Protein S-Palmitoylation in Synaptic Plasticity and Neurological Disorders: Potential and Limitations of Methods for Detection and Analysis

S-palmitoylation (S-PALM) is a lipid modification that involves the linkage of a fatty acid chain to cysteine residues of the substrate protein. This common posttranslational modification (PTM) is unique among other lipid modifications because of its reversibility. Hence, like phosphorylation or ubiquitination, it can act as a switch that modulates various important physiological pathways within the cell. Numerous studies revealed that S-PALM plays a crucial role in protein trafficking and function throughout the nervous system. Notably, the dynamic turnover of palmitate on proteins at the synapse may provide a key mechanism for rapidly changing synaptic strength. Indeed, palmitate cycling on postsynaptic density-95 (PSD-95), the major postsynaptic density protein at excitatory synapses, regulates the number of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and thus affects synaptic transmission. Accumulating evidence suggests a relationship between impairments in S-PALM and severe neurological disorders. Therefore, determining the precise levels of S-PALM may be essential for understanding the ways in which this PTM is regulated in the brain and controls synaptic dynamics. Protein S-PALM can be characterized using metabolic labeling methods and biochemical tools. Both approaches are discussed herein in the context of specific methods and their advantages and disadvantages. This review clearly shows progress in the field, which has led to the development of new, more sensitive techniques that enable the detection of palmitoylated proteins and allow predictions of potential palmitate binding sites. Unfortunately, one significant limitation of these approaches continues to be the inability to use them in living cells

An Interplay of S-Nitrosylation and Metal Ion Binding for Astrocytic S100B Protein.

Mammalian S100B protein plays multiple important roles in cellular brain processes. The protein is a clinically used marker for several pathologies including brain injury, neurodegeneration and cancer. High levels of S100B released by astrocytes in Down syndrome patients are responsible for reduced neurogenesis of neural progenitor cells and induction of cell death in neurons. Despite increasing understanding of S100B biology, there are still many questions concerning the detailed molecular mechanisms that determine specific activities of S100B. Elevated overexpression of S100B protein is often synchronized with increased nitric oxide-related activity. In this work we show S100B is a target of exogenous S-nitrosylation in rat brain protein lysate and identify endogenous S-nitrosylation of S100B in a cellular model of astrocytes. Biochemical studies are presented indicating S-nitrosylation tunes the conformation of S100B and modulates its Ca2+ and Zn2+ binding properties. Our in vitro results suggest that the possibility of endogenous S-nitrosylation should be taken into account in the further studies of in vivo S100B protein activity, especially under conditions of increased NO-related activity

Titration curves of PAR alone (diamonds) and in presence of either S100BSH (squares) or S100BSNO (triangles) proteins with ZnSO₄ measured in absorbance at 500 nm at 25°C.

<p>Experiments were performed at (A) 10 mM TES buffer, pH 7.2, 10 mM CaCl₂, 15 mM NaCl; (B) 10 mM TES buffer, pH 7.2, 15 mM NaCl and (C) 10 mM TES buffer, pH 7.2, 150 mM NaCl.</p

ITC-derived thermodynamic parameters for Zn²⁺ binding to S100BSH and S100BSNO protein dimer.

<p>ITC-derived thermodynamic parameters for Zn²⁺ binding to S100BSH and S100BSNO protein dimer.</p

Representative integrated heat plots obtained for ITC titrations of Zn²⁺ ions to S100BSH (yellow diamonds) and S100BSNO (blue diamonds) protein solutions.

<p>(A) Integrated heat plots for <i>holo</i> Ca²⁺-S100BSH and <i>holo</i> Ca²⁺-S100BSNO protein with ZnSO₄ in 10 mM TES buffer, pH 7.2, 15 mM NaCl. (B) Integrated heat plots for <i>apo</i> S100BSH and <i>apo</i> S100BSNO protein with ZnSO₄ in 10 mM TES buffer, pH 7.2, 15 mM NaCl. (C) Integrated heat plots for <i>apo</i> S100BSH and <i>apo</i> S100BSNO protein with ZnSO₄ in 10 mM TES buffer, pH 7.2, 150 mM NaCl.</p

H/D exchange results for S100BSH and S100BSNO as assessed by liquid chromatography combined with mass spectrometry (LC-MS).

<p>H/D exchange results for S100BSH and S100BSNO as assessed by liquid chromatography combined with mass spectrometry (LC-MS).</p

ITC-derived thermodynamic parameters for Ca²⁺ binding to S100BSH and S100BSNO protein monomers.

<p>ITC-derived thermodynamic parameters for Ca²⁺ binding to S100BSH and S100BSNO protein monomers.</p