Regulation Of Cellular Trafficking Research Articles

Targeting of Tetraspanin CD81 with Monoclonal Antibodies and Small Molecules to Combat Cancers and Viral Diseases

Tetraspanin CD81 plays major roles in cell-cell interactions and the regulation of cellular trafficking. This cholesterol-embarking transmembrane protein is a co-receptor for several viruses, including HCV, HIV-1 and Chikungunya virus, which exploits the large extracellular loop EC2 for cell entry. CD81 is also an anticancer target implicated in cancer cell proliferation and mobility, and in tumor metastasis. CD81 signaling contributes to the development of solid tumors (notably colorectal, liver and gastric cancers) and has been implicated in the aggressivity of B-cell lymphomas. A variety of protein partners can interact with CD81, either to regulate attachment and uptake of viruses (HCV E2, claudin-1, IFIM1) or to contribute to tumor growth and dissemination (CD19, CD44, EWI-2). CD81-protein interactions can be modulated with molecules targeting the extracellular domain of CD81, investigated as antiviral and/or anticancer agents. Several monoclonal antibodies anti-CD81 have been developed, notably mAb 5A6 active against invasion and metastasis of triple-negative breast cancer cells. CD81-EC2 can also be targeted with natural products (trachelogenin and harzianoic acids A-B) and synthetic compounds (such as benzothiazole-quinoline derivatives). They are weak CD81 binders but offer templates for the design of new compounds targeting the open EC2 loop. There is no anti-CD81 compound in clinical development at present, but this structurally well-characterized tetraspanin warrants more substantial considerations as a drug target.

Manufactured tissue-to-tissue barrier chip for modeling the human blood-brain barrier and regulation of cellular trafficking.

Microphysiological system or organ-on-a-chip technologies can replicate the key structure and function of 3D human tissues with higher reproducibility than less controllable 3D cell aggregate models, providing great potential to become advanced drug toxicity and efficacy test platforms alternative to animal models. However, these organ chip models remain to be manufactured and standardized in a highly reproducible manner for reliable drug screening and mechanism of action research. Herein, we present a manufactured form of 'micro-engineered physiological system-tissue barrier chip' called MEPS-TBC for the highly replicable modeling of the human blood-brain barrier (BBB) with a 3D perivascular space. The perivascular region was controlled by tunable aspiration, where human astrocytes reside in 3D, create a network, and communicate with human pericytes facing human vascular endothelial cells, thereby replicating the 3D BBB. The lower channel structure of MEPS-TBC was designed and optimized using a computational simulation to facilitate aspiration while maintaining multicellular construction. Our human BBB model of the 3D perivascular unit and the endothelium perfused by physiological shear stress secured significantly enhanced barrier function exhibiting greater TEER and lower permeability, compared to the only endothelial model, indicating that the cellular interactions between BBB cells significantly contribute to the BBB formation. Importantly, our BBB model showed the cellular barrier function for homeostatic trafficking regulation against inflammatory peripheral immune cells, as well as for molecular transport control across the BBB. We believe our manufactured chip technology will construct reliable and standardized organ-chip models for disease mechanism research and predictive drug screening.

MDia1 regulates breast cancer invasion by controlling membrane type 1-matrix metalloproteinase localization.

Mammalian diaphanous-related formin 1 (mDia1) expression has been linked with progression of malignant cancers in various tissues. However, the precise molecular mechanism underlying mDia1-mediated invasion in cancer cells has not been fully elucidated. In this study, we found that mDia1 is upregulated in invasive breast cancer cells. Knockdown of mDia1 in invasive breast cancer profoundly reduced invasive activity by controlling cellular localization of membrane type 1-matrix metalloproteinase (MT1-MMP) through interaction with microtubule tracks. Gene silencing and ectopic expression of the active form of mDia1 showed that mDia1 plays a key role in the intracellular trafficking of MT1-MMP to the plasma membrane through microtubules. We also demonstrated that highly invasive breast cancer cells possessed invasive activity in a 3D culture system, which was significantly reduced upon silencing mDia1 or MT1-MMP. Furthermore, mDia1-deficient cells cultured in 3D matrix showed impaired expression of the cancer stem cell marker genes, CD44 and CD133. Collectively, our findings suggest that regulation of cellular trafficking and microtubule-mediated localization of MT1-MMP by mDia1 is likely important in breast cancer invasion through the expression of cancer stem cell genes.

Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate.

The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.

Selective Expression of Huntingtin-associated Protein 1 in β-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington's disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy beta-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive beta-cells but not in the glucagon-immunoreactive alpha-cells and somatostatin immunoreactive delta-cells. In isolated rat pancreatic islets, approximately 80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in beta-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin.

Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are arrested in an immature and proliferating state. These leukemic cells can be grown in cell cultures and induced to terminal erythroid differentiation by a treatment with a specific chemical inducer such as N,N'-hexamethylene bisacetamide. MEL cells then re-enter their original erythroid program and differentiate along the erythroid pathway into non-dividing hemoglobin-rich cells resembling orthochromatophilic normoblasts. To deepen our understanding of the molecular mechanisms underlying and erythroid differentiation and leukemia we monitored changes in protein expression in differentiating MEL cells. In our effort to find new candidate proteins involved in the differentiation of MEL cells, we embraced a proteomic approach. Employing two-dimensional (2D) electrophoresis combined with mass spectrometry, we compared protein expression in non-induced MEL cells with MEL cells exposed to N,N'-hexamethylene bisacetamide for 48 h. From 700 proteins spots observed, 31 proteins were differentially expressed. We successfully identified 27 of the differentially expressed molecules by mass spectrometry (MALDI-MS). In addition to proteins involved in heme biosynthesis, protein metabolism, stress defense and cytoskeletal organization, we identified 3 proteins engaged in regulation of cellular trafficking and 7 proteins important for regulation of gene expression and cell cycle progression including 3 components of chromatin remodeling complexes. Many of the identified molecules are associated with erythroid differentiation or leukemia for the first time. To our knowledge, this is the first study applying a modern proteomic approach to the direct analysis of erythroid differentiation of leukemic cells.