Zhe Yang

Mammalian retromer complex contributes to multiple early endosome-associated trafficking pathways whose origins are dependent on which sorting nexin (SNX) they are complexed with. In an attempt to dissect out the contribution of individual retromer–SNX complexes, we examined the trafficking of EGFR in detail within a series of KO cell line models. We demonstrated that the depletion of retromer subunit Vps35 leads to decreased EGFR protein levels in resting cells with enhanced association of EGFR with lysosomal compartments. Compared to control cells, the addition of EGF to Vps35 KO cells resulted in a reduced rate of EGFR degradation; AKT activation and cell prolferation rates were elevated, while ERK activation remained relatively unchanged. These observations are consistent with a prolonged temporal association of EGFR within early endosomes due to the inefficiency of early endosome-associated protein trafficking pathways or organelle maturation due to retromer absence. We did not fully delineate the discrete contributions from retromer-associated SNXs to the phenotypes observed from retromer Vps35 depletion. While each of the knock-outs of SNX1/2, SNX3, or SNX27 promotes the enhanced association of EGFR with early endosomal compartments, only the decreased EGF-mediated EGFR degradation was observed in SNX1/2 dKO cells, while the enhanced AKT activation was only increased in SNX3 KO or SNX27 KO cells. Despite this, each of the knock-outs showed increased EGF-stimulated cell proliferation rates.

To date, mechanistic treatments targeting the initial cause of Parkinson's disease (PD) are limited due to the underlying biological cause(s) been unclear. Endosomes and their associated cellular homeostasis processes have emerged to have a significant role in the pathophysiology associated with PD. Several variants within retromer complex have been identified and characterised within familial PD patients. The retromer complex represents a key sorting platform within the endosomal system that regulates cargo sorting that maintains cellular homeostasis. In this review, we summarise the current understandings of how PD-associated retromer variants disrupt cellular trafficking and how the retromer complex can interact with other PD-associated genes to contribute to the disease progression.

Novel macrocyclic peptides are found that bind and modulate the function of the retromer membrane trafficking complex.

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.

The endosomal system is critical for the maintenance of intracellular homeostasis, and defects in this system are often linked to neurological disorders. The retromer complex is a critical coordinator of endosomal dynamics and has functional roles in multiple cellular processes through sorting cargoes from endosomes to the trans‐Golgi network (TGN) or to the plasma membrane. Mammalian retromer comprises a core Vps26‐Vps35‐Vps29 trimer and associates with a range of proteins to generate endosomal tubular‐vesicular carriers. Alterations in retromer function or its molecular organization have been a rising risk factor for Parkinson's disease (PD). Although genetic evidence has shown several variants within retromer in late‐onset PD cases, the exact molecular machineries by which retromer variants induce the development of PD are still not completely elucidated. In this Review, we will focus on the functional roles of retromer in neuronal health, summarize advances in defining the cellular pathological phenotype caused by retromer deficiency or PD‐linked retromer variants and discuss the potential clues of how retromer deregulation may contribute to PD pathogenesis.

To invade epithelial cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) induces macropinocytosis through the action of virulence factors delivered across the host cell membrane via a type III secretion system. We show that after docking at the plasma membrane S. Typhimurium triggers rapid recruitment of cytosolic SNX18, a SH3-PX-BAR domain sorting nexin protein, to the bacteria-induced membrane ruffles and to the nascent Salmonella-containing vacuole. SNX18 recruitment required the inositol-phosphatase activity of the Salmonella effector SopB and an intact phosphoinositide-binding site within the PX domain of SNX18, but occurred independently of Rho-GTPases Rac1 and Cdc42 activation. SNX18 promotes formation of the SCV from the plasma membrane by acting as a scaffold to recruit Dynamin-2 and N-WASP in a process dependent on the SH3 domain of SNX18. Quantification of bacteria uptake revealed that overexpression of SNX18 increased bacteria internalization, whereas a decrease was detected in cells overexpressing the phosphoinositide-binding mutant R303Q, the ΔSH3 mutant, and in cells where endogenous levels of SNX18 were knocked-down. This study identifies SNX18 as a novel target of SopB and suggests a mechanism where S. Typhimurium engages host factors via local manipulation of phosphoinositide composition at the site of invasion to orchestrate the internalization process.

Secretion of high molecular weight (HMW) adiponectin is dependent on post-translational modification (PTM) of conserved lysines in the collagenous domain. The present study aims to characterize the enzymes responsible for the PTM of conserved lysines which leads to HMW adiponectin secretion, and to define its significance in relation to obesity. Collagen beta (1-O) galactosyltransferase 1 (GLT25D1) was knocked down in HEK cells modified for the stable expression of adiponectin (adiponectin expressing human embryonic kidney cells, Adipo-HEK) as well as in Simpson Golabi-Behmel-Syndrome (SGBS) adipocytes. Knockdown of GLT25D1 caused a significant decrease in HMW adiponectin in Adipo-HEK cells with no change in total adiponectin. Knockdown in the SGBS cells caused an increase in lipid accumulation yet inhibited adipogenesis. Co-immunoprecipitation with adiponectin and mass spectrometry showed that adiponectin formed a protein complex with lysyl hydroxylase 3 (LH3) and GLT25D1. Transient overexpression of GLT25D1 showed that the intracellular retention of LH3 was dependent on GLT25D1. To determine whether changes in GLT25D1 were significant in obesity, mice were fed a standard chow or high-fat diet (HFD) for 5 weeks. GLT25D1 was significantly decreased in mice fed HFD which coincided with a decrease in HMW adiponectin. We conclude that GLT25D1 regulates HMW adiponectin secretion and lipid accumulation, consistent with changes in mice after high-fat feeding. These results suggest a novel function of GLT25D1 leading to decreased HMW adiponectin secretion in early obesity.

Many intracellular pathogens have evolved highly specialized mechanisms to isolate themselves from the host cell's innate immune response while still obtaining the necessary nutrients to survive. Salmonella utilizes type 3 secretion systems (T3SSs) to deliver bacterial proteins called effectors, across the encompassing Salmonella Containing vacuole (SCV) membrane, to subvert the host's membrane trafficking pathways and alter other cellular processes. The Salmonella Pathogenicity Island (SPI)‐2 effector SopD2 has recently been demonstrated to modulate multiple members of the Rab GTPase family such as Rab7, Rab8, Rab10, and Rab32 (D'Costa et al., , Cell Reports, 12:1508–18; Spano et al., , Cell Host & Microbe, 19:216–26). Here, we demonstrate the additional capacity of SopD2 to bind Rab34 and modulate its function. Our data indicate that depletion of Rab34 delays maturation of the SCV, and consequently, inhibits intracellular Salmonella enterica serotype typhimurium (S. typhimurium) growth. Interestingly, intracellular growth of the S. typhimurium lacking SopD2 was severely impaired in Rab34‐depleted cells, suggesting a compounding virulence effect. Overall this study reveals an additional member of the Rab GTPase family, Rab34, that is modulated by SopD2 and provides insight into its role in Salmonella biology.

Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95–Dlg–ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the β₂ adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor, lack these sequence determinants. Instead, we identified conserved sites of phosphorylation that substitute for acidic residues and dramatically enhance SNX27 interactions. This newly identified mechanism suggests a likely regulatory switch for PDZ interaction and protein transport by the SNX27–retromer complex. Defining this SNX27 binding code allowed us to classify more than 400 potential SNX27 ligands with broad functional implications in signal transduction, neuronal plasticity and metabolite transport.