Characterization of constitutive ER-phagy of excess membrane proteins.

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

A Rab5 GTPase module is important for autophagosome closure.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Distinct roles of TNF-related apoptosis-inducing ligand (TRAIL) in viral and bacterial infections: from pathogenesis to pathogen clearance.

INTRODUCTION: Apoptotic death of different cells observed during infection is thought to limit overwhelming inflammation in response to microbial challenge. However, the underlying apoptotic death mechanisms have not been well defined. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belonging to the TNF superfamily, which is involved not only in tumor growth suppression but in infection control and also in the regulation of both innate and adaptive immune responses. FINDINGS: In this review, we have summarized data of recent studies on the influence of the TRAIL/TRAIL receptor (TRAIL-R) system on the development of viral and bacterial infections. TRAIL may have a dual function in the immune system being able to kill infected cells and also to participate in the pathogenesis of multiple infections. Moreover, many pathogens have evolved mechanisms to manipulate TRAIL signaling thus increasing pathogen replication. CONCLUSION: Present data highlight an essential role for the TRAIL/TRAIL-R system in the regulation and modulation of apoptosis and show that TRAIL has distinct roles in pathogenesis and pathogen elimination. Knowledge of the factors that determine whether TRAIL is helpful or harmful supposes its potential therapeutic implications that are only beginning to be explored.

Acetylated derivative of glaucine inhibits joint inflammation in collagenase-induced arthritis.

CONTEXT: Osteoarthritis (OA) has become by far the most common joint disorder. A number of studies using OA animal models have explored the effects of agents that can modulate bone metabolism. OBJECTIVE: In the present study, we investigated the effect of acetylated derivative of plant alkaloid glaucine (ADG) on experimental OA in mice. MATERIALS AND METHODS: Arthritis was induced by two intraarticular (i.a.) injections of collaganase. Histopathological changes were observed through hematoxylin and eosine (H&E), safranin O and toluidine blue staining. Differentiation of bone marrow (BM) cells was evaluated by tartarate-resistant acid phosphatase (TRAP) assay. The expression of phospho-Janus kinase 2 (pJAK2) and phospho signal transducer and activator of transcription3 (pSTAT3) expression in the joints was determined by immunohistochemistry. RESULTS: We established that ADG significantly decreased cell infiltration (2.32 ± 0.14 versus 1.62 ± 0.13), cartilage loss (2.42 ± 0.12 versus 1.12 ± 0.10) and bone erosion (1.76 ± 0.13 versus 1.04 ± 0.14) in arthritic mice. It appeared that the substance inhibited in a dose-dependent manner osteoclast differentiation in vitro. ADG suppressed the expression of pJAK2 in the joint and partially affected the expression of pSTAT3. CONCLUSION: Present results suggest that ADG is a suitable candidate for further development as an anti-arthritic agent.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

Involvement of soluble receptor activator of nuclear factor-κB ligand (sRANKL) in collagenase-induced murine osteoarthritis and human osteoarthritis.

Joint destruction and excessive bone formation are associated with high expression of soluble receptor activator of nuclear factor-κB ligand (sRANKL). This study was undertaken to investigate the role of sRANKL in collagenase-induced osteoarthritis (CIOA) in mice and in patients with osteoarthritis (OA). The initial phase of CIOA was associated with severe proteoglycan depletion, decreased collagen density, and up-regulation of bone morphogenetic protein (BMP)-2. At the late stage of CIOA, bone remodeling was related with increased BMP2 and RANKL expression in the joints, high sRANKL, and decreased number of activated neutrophils in synovium. CIOA mice showed elevated plasma level of sRANKL but low RANKL expression on blood neutrophils. The percentage of RANKL-positive blood neutrophils was higher in patients with OA than in healthy individuals. Our data indicate that increased local and systemic levels of soluble RANKL might be indicative for OA disorders in mouse and human.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

Functional complement activity is decisive for the development of chronic synovitis, osteophyte formation and processes of cell senescence in zymosan-induced arthritis.

Synovial inflammation plays a critical role in the symptoms and structural progression of arthritis which leads to irreversible damage of the adjacent cartilage and bone. Activation of complement system is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis (RA). In this study, we show that the depletion of functional complement activity at the time of the initiation of zymosan-induced arthritis, significantly reduced the expression of TGF-beta1/3, BMP2 and pSmad2 and decreased the number of Sudan Black B positive cells in the synovium. Also, the excessive synthesis of proteoglycans and glycosaminoglycans was diminished. The appearance of apoptotic and senescent cells among the adherent bone marrow cells cultivated in vitro was not observed in complement depleted mice. Therefore, the lack of functional complement prevented the development of chronic synovitis, osteophyte formation and the generation of pathologic senescent arthritic cells.

Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock.

Sepsis remains a serious clinical problem despite continuous efforts to increase survival. Experimental animal models of sepsis are pointed to a great extent on blocking the activity of cytokines. A number of signal-transducing molecules are associated with the occurrence of excessive tissue inflammation. Through inhibition of tyrosine phosphorilation and thereby changing cell signaling, tyrosine kinase inhibitors can influence multiple inflammatory pathways. The purpose of the present investigation was to evaluate the effect of tyrosine kinase inhibitor tyrphostin AG490 in a mouse LPS-induced shock. Cytokine and chemokine blood levels were determined by ELISA assays. CD11b(+) Ly6C(+), CD3(+)CD69(+) and C5aR positive cell populations in the peritoneal exudate were detected by flow cytometry. The expression of iNOS and Signal Transducer and Activator of Transcription (STAT) in the liver were observed by immunohistochemistry. We found that tyrphostin AG490 inhibited Regulated upon activation normal T cell expressed and secreted (RANTES), IL-6 and IL-12 serum levels, decreased the number of CD11b(+)Ly6C(+) and CD3(+)CD69(+) subpopulations in the peritoneal exudate and prevented the decrease of cells expressing C5a receptor and TNF-alpha receptor. Tyrphostin ameliorated liver injury associated with suppressed iNOS, STAT3 and pSTAT3 expression. Our data suggest that tyrphostin AG490 diminished the degree of inflammation starting in peritoneal cavity and minimized liver dysfunction thus representing one approach for better outcome of sepsis conditions.

Tyrosine kinase inhibitor tyrphostin AG490 retards chronic joint inflammation in mice.

Tyrphostin AG490 is a Janus kinase (JAK) 2 inhibitor that is clinically used as an anticancer agent and is also effective in various models of inflammatory and autoimmune diseases. In this study, we examined the effects of tyrphostin AG490 on the development of collagenase-induced osteoarthritis (CIOA). Our results showed that tyrphostin-ameliorated cartilage and bone destructions. This effect was associated with decreased expression of signal transducers and activators of transcription 3 (STAT3), phosphorylated JAK2, Dickkopf homolog 1, and receptor activator of nuclear factor κB ligand (RANKL) in the joints of arthritic mice. Tyrphostin AG490 suppressed STAT3 phosphorylation and the expression of tumor necrosis factor-related apoptosis-inducing ligand and RANKL by synovial fluid cells. The drug inhibited RANKL-induced osteoclast differentiation in vitro. Molecules, such as tyrphostin AG490 that limit bone erosion and influence osteoclast generation, might have therapeutic utility in joint degenerative disorders.

The role of properdin in murine zymosan-induced arthritis.

Using properdin-deficient and wild-type mice, we have investigated the role of properdin in development and progression of zymosan-induced arthritis. At the initial phase of local, zymosan-induced inflammation, properdin-deficient and wild-type mice showed bone erosion, proteoglycan loss and cell infiltration. Compared to wild-type, properdin-deficient mice had reduced C5a and IL-6 but similar synovial TNF-alpha and sRANKL levels. Both groups showed a systemic immune response. Elevated IFN-gamma production and STAT1 signaling in splenocytes and a shift to Th1 response in popliteal lymph nodes were observed in properdin-deficient mice. Properdin-deficient mice had significantly less circulating zymosan-specific IgG antibodies than wild-type. In the chronic phase, the lack of properdin resulted in significant proteoglycan loss in the joints and lower cartilageneous STAT1 expression. The level of synovial C5a on day 30 was comparable in both groups, but C5aR staining was more apparent in the joints of properdin-deficient mice. Our data show that properdin is an important player in processes involved in inflammatory joint degradation.

Inhibition of zymosan-induced kidney dysfunction by tyrphostin AG-490.

BACKGROUND: Zymosan-induced shock has been associated with an increased production of pro-inflammatory cytokines and mediators, causing a generalized dysfunction of liver, lung and kidneys. Herein, we investigate the effects of tyrphostin AG-490 on the early inflammation and on the late renal injury provoked by zymosan injection. METHODS: Shock was induced by intraperitoneal injection of zymosan in a dose of 0.8-1.0 mg/g body weight in BALB/c mice and 0.8 mg/g body weight in SCID mice. Tyrphostin AG-490 was administered intraperitoneally in a dose of 5 mg/kg immediately after shock induction. Blood, peritoneal lavage and kidneys were collected at certain time points after zymosan injection. The levels of MIP-1alpha, RANTES, IL-6, IL-10, alpha1-antitrypsin and C5a in plasma were determined by ELISA. The number of IL-10-secreting cells in peritoneum was assayed by ELISPOT. Kidney function was monitored by measurement of urine/plasma creatinine levels and proteinuria. Histological assessment of renal injury was performed in a blinded fashion after hematoxylin/eosin staining. Immunohistochemistry analyses were used to evaluate the expression of C5aR, STAT1, STAT3 and the binding ability of IgGs in kidneys. RESULTS: Tyrphostin AG-490 attenuated the early phase of zymosan-induced shock via inhibition of MIP-1alpha, RANTES and C5a plasma levels and via elevation of IL-10 in plasma. The drug increased IL-10 production in peritoneum and the number of IL-10-secreting peritoneal cells. AG-490 was able to retain the time of coagulation and the level of alpha1-antitrypsin to normal values. At the late stage of shock, AG-490 decreased scores of tubular injury, cell infiltration and glomerular lesions in parallel with diminished creatinine plasma level and protein excretion. These beneficial effects of AG-490 were related to lowered levels of circulating IL-6, MIP-1alpha and C5a, and to inhibited expression of STAT1, STAT3 and C5aR in kidneys. The drug diminished the production of zymosan-specific IgG antibodies and hindered the glomeruli from IgGs recognition. CONCLUSION: Tyrphostin AG-490 reduced the magnitude of the initial inflammatory response in zymosan-induced shock and prevented the development of severe kidney dysfunction. Our data suggest that the drug might be used as a therapeutic approach in cases where shock is combined with acute renal injury.