Two distinct mechanisms target membrane proteins to the axonal surface

We have investigated the trafficking of two endogenous axonal membrane proteins, VAMP2 and NgCAM, in order to elucidate the cellular events that underlie their polarization. We found that VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2, selective delivery in the case of NgCAM.     

A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.     

On mechanisms of depression in pathways of presynaptic inhibition

We investigated inhibition of the N₁-component of the spinal cord dorsal potential (CDP) evoked by experimental stimulation of the n. peroneus in spinal cats. Stimulation was carried out following two conditioning stimuli administered at different time intervals to the same or different cutaneous nerves. The interval between the last conditioning stimulus and the experimental one remained constant (20 msec). It is demonstrated that there is no dependence between weakening of inhibitory action of the second conditioning stimulus and inhibition of the dorsal horn interneurons excited by it that generate the N₁-component of the CDP. It is hypothesized that mechanisms which act on the principle of negative feedback are present in the vincinity of the synaptic junctions of cutaneous afferent fibers with neurons of the substantia gelationsa, and that these mechanisms restrict the development of presynaptic inhibition during inflow of a series of afferent impulses into the cord.Dnepropetrovsk State University. Translated from Neirofiziologia, Vol. 1, No. 3, pp. 253–261, November–December, 1969.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Two distinct mechanisms of B-lymphocyte tolerance.

Based on the different characteristics of “pure” B-lymphocyte tolerance induction by polymeric and nonpolymeric antigens, it is proposed that there are two fundamentally distinct mechanisms by which mature B cells are tolerized: Polymeric antigens inhibit the expression of surface receptors irrespective of surface Ig isotype, whereas nonpolymeric antigens act by an unknown mechanism in which surface IgD is critically important in rendering cells “resistant” to tolerance induction. Further experiments to validate this hypothesis are highlighted.     

Two distinct pathways for cyclooxygenase-2 protein degradation

Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.     

Two distinct aggregation pathways in transthyretin misfolding and amyloid formation

Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2–5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.     

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

Receptor complexes cotransported via polarized endocytic pathways form clusters with distinct organizations

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.     

Two distinct integrin-mediated mechanisms contribute to apical lumen formation in epithelial cells

Background

Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of β1-integrins in epithelial polarization is well established but the significance of the accompanying α-subunits have not been analyzed in detail.

Principal Findings

Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) α2β1- and α6β4-integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) α3β1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing.

Significance

Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis during development and disease.     

Two distinct ubiquitin-proteolysis pathways in the fission yeast cell cycle

The SCF complex (Skp1-Cullin-1-F-box) and the APC/cyclosome (anaphase-promoting complex) are two ubiquitin ligases that play a crucial role in eukaryotic cell cycle control. In fission yeast F-box/WD-repeat proteins Pop1 and Pop2, components of SCF are required for cell-cycle-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor Rum1 and the S-phase regulator Cdc18. Accumulation of these proteins in pop1 and pop2 mutants leads to re-replication and defects in sexual differentiation. Despite structural and functional similarities, Pop1 and Pop2 are not redundant homologues. Instead, these two proteins form heterodimers as well as homodimers, such that three distinct complexes, namely SCFPop1/Pop1, SCFPop1/Pop2 and SCFPop2/Pop2, appear to exist in the cell. The APC/cyclosome is responsible for inactivation of CDK/cyclins through the degradation of B-type cyclins. We have identified two novel components or regulators of this complex, called Apc10 and Ste9, which are evolutionarily highly conserved. Apc10 (and Ste9), together with Rum1, are required for the establishment of and progression through the G1 phase in fission yeast. We propose that dual downregulation of CDK, one via the APC/cyclosome and the other via the CDK inhibitor, is a universal mechanism that is used to arrest the cell cycle at G1.     

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid.

Results of in vitro experiments have suggested the existence of at least three pathways by which nuclear-encoded proteins are targeted to the chloroplast thylakoid membrane. However, few components of the targeting machinery have been identified and the relationship between the three pathways is not clear. To investigate mechanisms underlying thylakoid protein targeting, we identified nuclear mutations in maize that cause targeting defects. We found two mutations, tha1 and hcf106, that disrupt the localization of different sets of proteins to the thylakoid lumen. The tha1 mutation interferes with the targeting of one chloroplast-encoded protein, cytochrome f, and three nuclear-encoded proteins, plastocyanin, the psaF gene product and the 33 kDa subunit of the oxygen-evolving complex. The hcf106 mutation interferes with the targeting of the 16 and 23 kDa subunits of the oxygen-evolving complex. The tha1 and hcf106 phenotypes provide the first in vivo evidence supporting the existence of two distinct thylakoid-targeting pathways. Their phenotypes also provide evidence that one chloroplast-encoded protein, cytochrome f, engages the 'tha1' pathway, indicating that nuclear- and chloroplast-encoded proteins can be targeted via common machinery.     

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.     

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.     

Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease

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Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.