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TRPML Subfamily of Endolysosomal Channels
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Nicholas E. Karagas, Morgan A. Rousseau, Kartik Venkatachalam
To overcome the limitations of LysoTracker, co-labelling with specific-protein markers is also required. For instance, Rab5 and Rab7, which localize to the early- and late-endosomes, respectively, may be used as markers. Other markers of early endosome include HRS and EEA1, and late-endosomes/lysosomes include proteins such as LAMP1 and LAMP3 (Manzoni et al., 2004; Thompson et al., 2007; Venkatachalam et al., 2006). Lysosomes can be visualized using fluorescently tagged substrates for lysosomal enzymes such as cathepsin (e.g. Magic Red cathepsin L substrate) (Johnson et al., 2016). Autophagosomes are detected by labelling with fluorescently tagged Atg proteins or LC3, whereas amphisomes, which are generated by the fusion of autophagosomes and endosomes (Tanida et al., 2008), can be identified by appropriate co-labelling. In addition to these fluorescent techniques, autophagosomes, endosomes, amphisomes, and lysosomes can be effectively discriminated by electron microscopy, and have been shown to accumulate in cells lacking TRPMLs (Wong et al., 2012). Unfortunately, the absence of effective antibodies against TRPML proteins has prevented visualization of natively expressed proteins, which can be circumvented by examining the expression of ectopic expression of tagged TRPMLs.
Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold
Published in mAbs, 2023
Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, Hervé Nozach
We decided to generate structural and 3D modeling data to distinguish positions directly involved in the epitope from those affecting the overall conformation of the antigen and its folding, and ultimately refine the epitopes. We first solved the crystallographic structure of the complex between Fab B and an aglycosylated form of the first luminal domain of human LAMP-1 (Figure 4a). This domain adopts the same overall β-prism fold as murine LAMP-123 and DC-Lamp3.27 Most of the interaction between Fab B and LAMP-1 is mediated by amino acids in the heavy chain complementarity-determining regions (CDRs). Briefly, loop 82–86 of LAMP-1 interacts with CDRH1 and the FR3 loop from the Fab heavy chain. Loop 106–109 interacts with all heavy chain CDRs and loop 149–151 is in contact with CDRH3 and CDRL1. Lastly, loop 178–187 contacts both CDRH1 and CDRH3, along with CDRL1 and CDRL2. All LAMP-1 amino acids at the interface, i.e., at less than 4.5 Å from the Fab molecule are represented in yellow in Figure 4a and constitute what might be termed the ‘structural epitope’.
Dissecting the heterogeneity of the microenvironment in primary and recurrent nasopharyngeal carcinomas using single-cell RNA sequencing
Published in OncoImmunology, 2022
Wen-Sa Peng, Xin Zhou, Wen-Bin Yan, Yu-Jiao Li, Cheng-Run Du, Xiao-Shen Wang, Chun-Ying Shen, Qi-Feng Wang, Hong-Mei Ying, Xue-Guan Lu, Ting-Ting Xu, Chao-Su Hu
Identified in multiple cancers using scRNA-seq, LAMP3+ DCs are considered as a subset of regulatory or tolerogenic DCs.61–64LAMP3+ DCs were found to shape the immunosuppressive ecosystem in rNPC samples in our study. In HCC, the LAMP3+ DC level was positively correlated with the infiltration of exhausted CD8+ T cells and Tregs.62 In mouse models, LAMP3+ DCs can stimulate the clonal expansion of Th1-like cells.65 These findings were consistent with the elevated exhausted CTL features, and Treg and Th1 activity in rNPC, which showed promising treatment effects by targeting LAMP3+ DCs through blocking CCL19 chemotaxis.62 Similarly, M2-polarized macrophage signatures in rNPC resemble the findings of the C1Q+ TAM subset, which showed polarized phagocytosis, and antigen processing and presentation.62,65–67 Cell communication and TCGA enrichment in CRC suggested that C1Q+TAMs could recruite and regulate CD8+ exhausted T cells, Tregs, and Th1-like cells,65 which were consistent with features we found in recurrent samples. Transforming this macrophage subtype and turning M2 polarized TAM into M1 can be of potential value in future treatment of rNPC.
Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation
Published in OncoImmunology, 2020
Jay S. Wunder, Minji J. Lee, Junghyun Nam, Beatrice Y. Lau, Brendan C. Dickson, Dushanthi Pinnaduwage, Shelley B. Bull, Peter C. Ferguson, Andrew Seto, Nalan Gokgoz, Irene L. Andrulis
Ontology analysis of the biological processes of the 41 differentially expressed genes, including CD247, LAMP3 and a number of immunoglobulin genes in UPS and OSA (Figure 8, Supplementary Table 11) indicated the involvement of an adaptive immune response and humoral immune response. LAMP3 has been shown to be highly expressed in dendritic cells during cell differentiation and maturation.38 LAMP3 positive dendritic cells were found to be correlated with density of tumor HEVs, lymphocyte infiltration and favorable outcome in breast cancer.39 Further characterization of the role of dendritic cells and LAMP3 will also be also important for the development of efficient therapeutic strategies in sarcoma tumors. These results require further study, but may suggest additional avenues for combined therapy for some sarcomas. PD-L1 expression in UPS is most likely part of an adaptive response to the ongoing inflammatory immune attack by TILs15,16 and may represent an effective anti-tumoral immune response leading to improved clinical outcomes.