Role of GARP Vesicle Tethering Complex in Golgi Physiology.

The Golgi associated retrograde protein complex (GARP) is an evolutionarily conserved component of Golgi membrane trafficking machinery that belongs to the Complexes Associated with Tethering Containing Helical Rods (CATCHR) family. Like other multisubunit tethering complexes such as COG, Dsl1, and Exocyst, the GARP is believed to function by tethering and promoting fusion of the endosome-derived small trafficking intermediate. However, even twenty years after its discovery, the exact structure and the functions of GARP are still an enigma. Recent studies revealed novel roles for GARP in Golgi physiology and identified human patients with mutations in GARP subunits. In this review, we summarized our knowledge of the structure of the GARP complex, its protein partners, GARP functions related to Golgi physiology, as well as cellular defects associated with the dysfunction of GARP subunits.

Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines.

The Golgi-associated retrograde protein (GARP) complex is proposed to tether endosome-derived transport vesicles, but the exact function and mechanism of GARP action are not completely understood. To uncover the GARP function in human cells, we employ CRISPR/Cas9 strategy and knock out (KO) the unique VPS54 subunit of the GARP complex. In this chapter, we describe the detailed method of generating CRISPR/Cas9-mediated VPS54-KO in hTERT-RPE1 cells, rescue of resulting KO cells with stable near-endogenous expression of myc-tagged VPS54, and validation of KO and rescued (KO-R) cells using Western blot and immunofluorescence approaches. This approach is helpful in uncovering new functions of the GARP and other vesicle tethering complexes.

GARP dysfunction results in COPI displacement, depletion of Golgi v-SNAREs and calcium homeostasis proteins.

Golgi-associated retrograde protein (GARP) is an evolutionary conserved heterotetrameric protein complex that tethers endosome-derived vesicles and is vital for Golgi glycosylation. Microscopy and proteomic approaches were employed to investigate defects in Golgi physiology in RPE1 cells depleted for the GARP complex. Both cis and trans-Golgi compartments were significantly enlarged in GARP-knock-out (KO) cells. Proteomic analysis of Golgi-enriched membranes revealed significant depletion of a subset of Golgi residents, including Ca2+ binding proteins, enzymes, and SNAREs. Validation of proteomics studies revealed that SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, were significantly depleted in GARP-KO cells. Finding that GARP-KO is more deleterious to Golgi physiology than deletion of GARP-sensitive v-SNAREs, prompted a detailed investigation of COPI trafficking machinery. We discovered that in GARP-KO cells COPI is significantly displaced from the Golgi and partially relocalized to the ER-Golgi intermediate compartment (ERGIC). Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1, and BIG1 are also relocated to off-Golgi compartments. We propose that the dysregulation of COPI machinery, along with the depletion of Golgi v-SNAREs and alteration of Golgi Ca2+ homeostasis, are the major driving factors for the depletion of Golgi resident proteins, structural alterations, and glycosylation defects in GARP deficient cells.

Getting Sugar Coating Right! The Role of the Golgi Trafficking Machinery in Glycosylation.

The Golgi is the central organelle of the secretory pathway and it houses the majority of the glycosylation machinery, which includes glycosylation enzymes and sugar transporters. Correct compartmentalization of the glycosylation machinery is achieved by retrograde vesicular trafficking as the secretory cargo moves forward by cisternal maturation. The vesicular trafficking machinery which includes vesicular coats, small GTPases, tethers and SNAREs, play a major role in coordinating the Golgi trafficking thereby achieving Golgi homeostasis. Glycosylation is a template-independent process, so its fidelity heavily relies on appropriate localization of the glycosylation machinery and Golgi homeostasis. Mutations in the glycosylation enzymes, sugar transporters, Golgi ion channels and several vesicle tethering factors cause congenital disorders of glycosylation (CDG) which encompass a group of multisystem disorders with varying severities. Here, we focus on the Golgi vesicle tethering and fusion machinery, namely, multisubunit tethering complexes and SNAREs and their role in Golgi trafficking and glycosylation. This review is a comprehensive summary of all the identified CDG causing mutations of the Golgi trafficking machinery in humans.

The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery.

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-knockout (KO) cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].

Critical role of tristetraprolin and AU-rich element RNA-binding protein 1 in the suppression of cancer cell growth by globular adiponectin.

Adiponectin exhibits potent antitumor activities. Herein, we examined the molecular mechanisms underlying suppression of tumor growth by globular adiponectin (gAcrp). We demonstrated that gAcrp suppressed B-cell lymphoma 2 (Bcl-2) expression, an anti-apoptotic gene, by inducing its mRNA destabilization, which was accompanied with a decrease in cell viability and increased caspase-3 activity in hepatic cancer cells. In addition, gAcrp increased expression of tristetraprolin (TTP) and AU-rich element RNA-binding protein 1 (AUF1), which are mRNA stability regulatory proteins. Moreover, gAcrp-induced suppression of Bcl-2 expression was abrogated by knockdown of TTP or AUF1. These data indicate that gAcrp induces apoptosis of hepatic cancer cells by TTP- and AUF1-mediated Bcl-2 mRNA destabilization, and further suggest that TTP and AUF1 are novel targets mediating the antitumor activity of adiponectin.

Globular adiponectin protects hepatocytes from tunicamycin-induced cell death via modulation of the inflammasome and heme oxygenase-1 induction.

Endoplasmic reticulum (ER) stress, which is defined as the accumulation of unfolded or misfolded proteins in the ER, triggers cellular dysfunction and eventually leads to cell death. In particular, excessive and prolonged ER stress is closely related with hepatic injury. Adiponectin, a hormone predominantly produced by adipose tissue, is known to possess potent hepatoprotective properties and exhibits a cytoprotective effect in response to chronic ER stress. However, the underlying mechanisms are not clearly understood. In the present study, we examined the protective effect of globular adiponectin (gAcrp) on tunicamycin-induced cell death and further investigated its potential underlying mechanisms in rat hepatocytes. Herein, we found that treatment with gAcrp inhibited tunicamycin-induced cell death, decreased lactate dehydrogenase release (marker of pyroptotic cell death), and suppressed caspase activation; clearly indicating that gAcrp protects liver cells from ER stress. Interestingly, gAcrp prevented the tunicamycin-induced activation of the inflammasome, a key platform involved in the production of inflammatory cytokines that induces pyroptosis, determined by suppression of interleukin-1ß (IL-1ß) maturation, apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) speck formation, and caspase-1 activation. Moreover, we showed that suppression of the inflammasome activation by gAcrp was mediated via modulation of reactive oxygen species (ROS) production, particularly inhibition of NADPH oxidase. In addition, inhibition of heme oxygenase-1 (HO-1) signaling by pretreatment with SnPP, a pharmacological inhibitor of HO-1, or transfection with an siRNA targeting HO-1, abrogated the protective effects of gAcrp against tunicamycin-induced cell death and abolished the suppressive effect on the inflammasome activation, demonstrating that HO-1 signaling plays a crucial role in the protective effect of gAcrp against tunicamycin-induced damage in liver cells. Taken together, these results indicate that gAcrp protects liver cells from ER stress by modulating inflammasomes activation, at least in part, via HO-1 signaling-dependent mechanisms.

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study.

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.