Fibroblasts from idiopathic Parkinson’s disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2

Lysosomal dysfunction is a central pathway associated with Parkinson’s disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.

Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

Comprehensive mapping of immune perturbations associated with severe COVID-19

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified extensive induction and activation of multiple immune lineages, including T cell activation, oligoclonal plasmablast expansion, and Fc and trafficking receptor modulation on innate lymphocytes and granulocytes, that distinguished severe COVID-19 cases from healthy donors or SARS-CoV-2-recovered or moderate severity patients. We found the neutrophil to lymphocyte ratio to be a prognostic biomarker of disease severity and organ failure. Our findings demonstrate broad innate and adaptive leukocyte perturbations that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.

Manganese-induced trafficking and turnover of GPP130 is mediated by sortilin

Elevated, nontoxic doses of manganese (Mn) protect against Shiga toxin-1–induced cell death via down-regulation of GPP130, a cycling Golgi membrane protein that serves as an endosome-to-Golgi trafficking receptor for the toxin. Mn binds to GPP130 in the Golgi and causes GPP130 to oligomerize/aggregate, and the complexes are diverted to lysosomes. In fact, based on experiments using the self-interacting FM domain, it appears generally true that aggregation of a Golgi protein leads to its lysosomal degradation. How such oligomers are selectively sorted out of the Golgi is unknown. Here we provide evidence that Mn-induced exit of GPP130 from the trans-Golgi network (TGN) toward lysosomes is mediated by the sorting receptor sortilin interacting with the lumenal stem domain of GPP130. In contrast, FM-induced lysosomal trafficking of the Golgi protein galactosyltransferase was sortilin independent and occurred even in the absence of its native lumenal domain. Thus sortilin-dependent as well as sortilin-independent sorting mechanisms target aggregated Golgi membrane proteins for lysosomal degradation.

Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin

Manganese (Mn) protects cells against lethal doses of purified Shiga toxin by causing the degradation of the cycling transmembrane protein GPP130, which the toxin uses as a trafficking receptor. Mn-induced GPP130 down-regulation, in addition to being a potential therapeutic approach against Shiga toxicosis, is a model for the study of metal-regulated protein sorting. Significantly, however, the mechanism by which Mn regulates GPP130 trafficking is unknown. Here we show that a transferable trafficking determinant within GPP130 bound Mn and that Mn binding induced GPP130 oligomerization in the Golgi. Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes. Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn. These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.

All-trans-Retinoic Acid Imprints Expression of the Gut-Homing Marker α4β7 while Suppressing Lymph Node Homing of Dendritic Cells

ABSTRACTTissue-directed trafficking of dendritic cells (DCs) as natural adjuvants and/or direct vaccine carriers is highly attractive for the next generation of vaccines and immunotherapeutics. Since these types of studies would undoubtedly be first conducted using nonhuman primate models, we evaluated the ability of all-trans-retinoic acid (ATRA) to induce gut-homing α4β7 expression on rhesus macaque plasmacytoid and myeloid DCs (pDCs and mDCs, respectively). Induction of α4β7 occurred in both a time-dependent and a dose-dependent manner with up to 8-fold increases for mDCs and 2-fold increases for pDCs compared to medium controls. ATRA treatment was also specific in inducing α4β7 expression, but not expression of another mucosal trafficking receptor, CCR9. Unexpectedly, upregulation of α4β7 was associated with a concomitant downregulation of CD62L, a marker of lymph node homing, indicating an overall shift in the trafficking repertoire. These same phenomena occurred with ATRA treatment of human and chimpanzee DCs, suggesting a conserved mechanism among primates. Collectively, these data serve as a first evaluation forex vivomodification of primate DC homing patterns that could later be used in reinfusion studies for the purposes of immunotherapeutics or mucosa-directed vaccines.