ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner

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Efficient preparation of glycoprotein hormones lacking an alpha-subunit oligosaccharide

The oligosaccharide on alpha-subunit loop 2 (alpha 2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha 2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F). Re-assembly of human choriogonadotropin, human follitropin, and bovine lutropin occurred rapidly and efficiently following removal of the alpha 2 oligosaccharide by PNGase F. Consequently, virtually all heterodimers formed in the presence of this enzyme lacked this oligosaccharide. These findings support the notion that heterodimer assembly in vitro occurs by a threading mechanism that is impeded by the presence of the alpha 2 oligosaccharide. This procedure should facilitate the study of glycoprotein hormone structure and function.     

Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via AKT and MAPK pathways

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to promote apoptosis in cancer cells. However, the role of EGCG in endothelial cells following ischemia/reperfusion (I/R) injury remains unclear. In the present study, we investigated the mechanisms by which EGCG enhances I/R-induced cell growth inhibition and apoptosis in human umbilical vein endothelial cells (HUVECs). Our results showed that EGCG treatment caused cell proliferation inhibition during I/R injury, and this effect was associated with increased p27 and p21 levels and reduced cyclin D1 level. Moreover, treatment of cells with EGCG resulted in increase of caspase-3 and Bax and decrease of Bcl-2, enhancing I/R-induced apoptosis. Interestingly, EGCG decreased I/R-induced phosphorylation of AKT and its downstream substrates Foxo1 and Foxo3a and ERK1/2. In contrast, EGCG increased JNK1/2 and c-Jun phosphorylation. Furthermore, both wortamannin (PI3K inhibitor) and U0126 (MEK1/2 inhibitor) markedly enhanced EGCG-induced apoptosis during I/R, whereas SP600125 (JNK inhibitor) attenuated the action of EGCG. Taken together, our study for the first time suggest that EGCG is able to enhance growth arrest and apoptosis of HUVECs during I/R injury, at least in part, through inhibition of AKT and ERK1/2 and activation of JNK1/2 signaling pathways.     

Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.     

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of alpha-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a "zipper-like" sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the beta-subunit cystine knot, alpha-subunit loop 2, and the small seatbelt loop. The resulting intersubunit beta-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.     

Only a portion of the small seatbelt loop in human choriogonadotropin appears capable of contacting the lutropin receptor

Twenty residues of the human choriogonadotropin (hCG) beta-subunit that are wrapped around alpha-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the alpha-subunit C terminus (alphaCT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of alphaCT residue alphaSer-92 and cysteines substituted for loop residues betaArg-94, betaArg-95, or betaSer-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the alphaCT was cross-linked to cysteines in place of residues betaArg-95 and betaAsp-99, but not to cysteines in place of residues betaArg-94, betaSer-96, or betaThr-97. This suggested that the side chains of residues betaArg-95 and betaAsp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small alphaCT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue betaAsp-99.     

Model of glycoprotein hormone receptor ligand binding and signaling

Studies described here were initiated to develop a model of glycoprotein hormone receptor structure and function. We found that the region that links the lutropin receptor leucine-rich repeat domain (LRD) to its transmembrane domain (TMD) has substantial roles in ligand binding and signaling, hence we term it the signaling specificity domain (SSD). Theoretical considerations indicated the short SSDs in marmoset lutropin and salmon follitropin receptors have KH domain folds. We assembled models of lutropin, follitropin, and thyrotropin receptors by aligning models of their LRD, TMD, and shortened SSD in a manner that explains how substitutions in follitropin and thyrotropin receptors distant from their apparent ligand binding sites enable them to recognize lutropins. In these models, the SSD is parallel to the concave surface of the LRD and makes extensive contacts with TMD outer loops 1 and 2. The LRD appears to contact TMD outer loop 3 and a few residues in helices 1, 5, 6, and 7. We propose that signaling results from contacts of the ligands with the SSD and LRD that alter the LRD, which then moves TMD helices 6 and 7. The positions of the LRD and SSD support the notion that the receptor can be activated by hormones that dock with these domains in either of two different orientations. This would account for the abilities of some ligands and ligand chimeras to bind multiple receptors and for some receptors to bind multiple ligands. This property of the receptor may have contributed significantly to ligand-receptor co-evolution.     

The structural basis for tight control of PP2A methylation and function by LCMT-1

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Highlights? LCMT-1 makes extensive contacts to PP2A active site for methylation of PP2A tail ? PP2A methylation is stimulated by phosphatase activation, hampered by inactivation ? LCMT-1 would facilitate efficient transition of activated PP2A to holoenzymes ? A high-affinity dnLCMT-1 mutant attenuates the cell cycle without causing cell death     

Proteasome-dependent inactivation of Akt is essential for 12-O-tetradecanoylphorbol 13-acetate-induced apoptosis in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. Here we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on SMC apoptosis. We found that TPA treatment induced SMC apoptosis through the rapid downregulation of Akt phosphorylation. The inhibition of Akt activation by TPA was markedly reduced by inhibitors of protein phosphatase 2A and proteasome. Moreover, TPA promoted the ubiquitination of p-Akt, whereas inhibition of TPA-induced PKC activation suppressed the downregulation and ubiquitination of p-Akt. Taken together, these results demonstrate that TPA triggers inactivation of Akt, at least in part, through PKC and Ubiquitin–proteasome degradation, thereby contributing to SMC apoptosis.