Protein quality control in the early secretory pathway

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.     

Dissecting glycoprotein quality control in the secretory pathway

In the early secretory pathway, asparagine-linked glycosylation facilitates the conformational maturation of diverse polypeptides by promoting their physical engagement with the glycoprotein-folding machinery. Misfolded glycoproteins are selectively eliminated from the endoplasmic reticulum by a stringent process of conformation-based quality control. Recent studies indicate that a small ensemble of oligosaccharide-processing enzymes and lectins use the asparagine-linked appendage to orchestrate the selective disposal of numerous transport-defective glycoproteins from the early secretory pathway. The glycan-based disposal system functions as an evolutionarily conserved terminal checkpoint in eukaryote genome expression. That the mechanisms by which glycoprotein substrates are recruited for degradation diverge at the level of signal recognition reflects a previously unappreciated component of cellular differentiation in higher eukaryotes.     

Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.     

Quality control in the secretory assembly line

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway.     

Quality control in the yeast secretory pathway: a misfolded PMA1 H+-ATPase reveals two checkpoints

The yeast plasma-membrane H(+)-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H(+)-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.     

Oligosaccharyl transferase: gatekeeper to the secretory pathway

Oligosaccharyl transferase is part of the macromolecular machinery that processes nascent proteins in the endoplasmic reticulum. The enzyme is highly conserved, catalyzes the initial step in the biosynthesis of N-linked glycoproteins and acts as a 'gatekeeper' for the secretory pathway. As more proteins associated with oligosaccharyl transferase are identified, the intricacies of the enzyme and the relationship with other proteins in the lumen of the endoplasmic reticulum are starting to be unraveled.     

Lectin control of protein folding and sorting in the secretory pathway

Glycan moieties are essential for folding, sorting and targeting of glycoproteins through the secretory pathway to various cellular compartments. The molecular mechanisms that underlie these processes, however, are only now coming to light. Recent crystallographic and NMR studies of proteins located in the endoplasmic reticulum (ER), Golgi complex and ER-Golgi intermediate compartment have illuminated their roles in glycoprotein folding and secretion. Calnexin and calreticulin, both ER-resident proteins, have lectin domains that are crucial for their function as chaperones. The crystal structure of the carbohydrate-recognition domain of ER-Golgi intermediate compartment (ERGIC)-53 complements the biochemical and functional characterization of the protein, confirming that a lectin domain is essential for the role of this protein in sorting and transfer of glycoproteins from the ER to the Golgi complex. The lectin domains of calnexin and ERGIC-53 are structurally similar, although there is little primary sequence similarity. By contrast, sequence similarity between ERGIC-53 and vesicular integral membrane protein (VIP36), a Golgi-resident protein, leaves little doubt that a similar lectin domain is central to the transport and/or sorting functions of VIP36. The theme emerging from these studies is that carbohydrate recognition and modification are central to mediation of glycoprotein folding and secretion.     

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Peptide aptamers expressed in the secretory pathway interfere with cellular PrPSc formation

Prion diseases are rare and obligatory fatal neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrPSc) of the host-encoded prion protein (PrPc). Prophylactic and therapeutic regimens against prion diseases are very limited. To extend such strategies we selected peptide aptamers binding to PrP from a combinatorial peptide library presented on the Escherichia coli thioredoxin A (trxA) protein as a scaffold. In a yeast two-hybrid screen employing full-length murine PrP (aa 23-231) as a bait we identified three peptide aptamers that reproducibly bind to PrP. Treatment of prion-infected cells with recombinantly expressed aptamers added to the culture medium abolished PrPSc conversion with an IC50 between 350 and 700 nM. For expression in eukaryotic cells, peptide aptamers were fused to an N-terminal signal peptide for entry of the secretory pathway. The C terminus was modified by a glycosyl-phosphatidyl-inositol-(GPI) anchoring signal, a KDEL retention motif and the transmembrane and cytosolic domain of LAMP-I, respectively. These peptide aptamers retained their binding properties to PrPc and, depending on peptide sequence and C-terminal modification, interfered with endogenous PrPSc conversion upon expression in prion-infected cells. Notably, infection of cell cultures could be prevented by expression of KDEL peptide aptamers. For the first time, we show that trxA-based peptide aptamers can be targeted to the secretory pathway, thereby not losing the affinity for their target protein. Beside their inhibitory effect on prion conversion, these molecules could be used as fundament for rational drug design.     

Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in the seminiferous epithelium. In this study, we crossed Ppp6c^F/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.Subject terms: Spermatogenesis, Infertility     

Metalloregulation of triple helix formation by control of the loop conformation

The flexible polypyridine ligand, 2,2':6',2(')-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu(2+), Zn(2+), and Fe(2+)), in particular Cu(2+) ions. This ion destabilized one of the triplexes by 4.2 kcalmol(-1) or made the triplex formation constant less than 1/10(3) at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

Mammary epithelial-specific deletion of the focal adhesion kinase gene leads to severe lobulo-alveolar hypoplasia and secretory immaturity of the murine mammary gland

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.     

Sorting of an exocrine secretory protein to the regulated secretory pathway in endocrine cells

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.     

Glutathione is required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway

The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum.     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP₇ and IP₈; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP₄ and IP₅. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.