Primary cilia are antenna-like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi-derived vesicles to the pericentrosome, a process requiring Rab11-mediated recruitment of Rabin8, a GDP-GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser-272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8-/Rab11-containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP-bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho-mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U ,C7U ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.

Zinc ions (Zn2+) are imported into the early secretory pathway by Golgi-resident transporters, but their handling and functions are not fully understood. Here, we show that Zn2+ binds with high affinity to the pH-sensitive chaperone ERp44, modulating its localization and ability to retrieve clients like Ero1α and ERAP1 to the endoplasmic reticulum (ER). Silencing the Zn2+ transporters that uptake Zn2+ into the Golgi led to ERp44 dysfunction and increased secretion of Ero1α and ERAP1. High-resolution crystal structures of Zn2+-bound ERp44 reveal that Zn2+ binds to a conserved histidine-cluster. The consequent large displacements of the regulatory C-terminal tail expose the substrate-binding surface and RDEL motif, ensuring client capture and retrieval. ERp44 also forms Zn2+-bridged homodimers, which dissociate upon client binding. Histidine mutations in the Zn2+-binding sites compromise ERp44 activity and localization. Our findings reveal a role of Zn2+ as a key regulator of protein quality control at the ER-Golgi interface.

Synthetic insulin analogues with al ong lifetime are current drug targets for the therapyo fd iabetic patients.T he replacement of the interchain disulfide with adiselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U A ,C7U B ]v ariant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A-and Bchains,r espectively). In ab uffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited ab ioactivity comparable to that of BPIns.I nterestingly, degradation of Se-Ins with IDE was significantly decelerated (t 1/2 % 8hvs. % 1hfor BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.Insulin, as mall globular protein (5.8 kDa), comprises two peptide chains,t he A-chain (Ins-A, 21 amino acid residues) and B-chain (Ins-B,3 0a mino-acid residues). Then ative structure in am onomeric active state is stabilized by two interchain disulfide bridges,Cys A7 -Cys B7 and Cys A20 -Cys B19 ,in addition to one intrachain disulfide linkage,C ys A6 -Cys A11 . [1] Considerable efforts have been directed toward development of various insulin analogues [2] which imitate either bolus secretion of insulin for expeditiously reducing postprandial blood glucose levels [3] or basal secretion of insulin to control the glucose level for an entire day. [4] Thel atter long-acting analogues have been designed so that insulin forms infusible precipitates or soluble oligomers (hexamer or dihexamer) under physiological conditions and slowly releases active insulin monomers.In contrast, the insulin-degrading enzyme (IDE) is ap ossible alternative target for diabetes therapy.I DE, which is involved in clearance of insulin and amyloid b (Ab), [5] is found in the liver and kidneys.Recent research has revealed that synthetic IDE inhibitors increase circulation of insulin by preventing its degradation in the liver,t hus resulting in improvement of the postprandial glucose tolerance. [6] However,other research suggests that IDE inhibitors could induce accumulation of Ab in the brain, [7] and would lead to Ab-mediated cognitive impairment. Hence,the design of long-lasting insulin analogues resistant against IDE would be desirable. [8] In this study,wehave attempted anew approach to alonglasting insulin analogue by exploiting the unique chemical properties of adiselenide bond. Namely,i ntroduction of two juxtaposed selenium atoms to the insulin analogue could lead to ah igher kinetic and thermodynamic stability than that of the wild-type without affecting the bioactivity.T his new strategy is based primarily on the higher rotational barrier of aSe À Se bond (ca. 4kcal mol À1 )than that of an S À Sbond (ca. 3kcal mol À1 ), [9] and se...

Although many Zn 2+ fluorescent probes have been developed, there remains a lack of consensus on the labile Zn 2+ concentrations ([Zn 2+ ]) in several cellular compartments, as the fluorescence properties and zinc affinity of the fluorescent probes are greatly affected by the pH and redox environments specific to organelles. In this study, we developed two turn-on-type Zn 2+ fluorescent probes, namely, ZnDA-2H and ZnDA-3H, with low pH sensitivity and suitable affinity (K d = 5.0 and 0.16 nM) for detecting physiological labile Zn 2+ in various cellular compartments, such as the cytosol, nucleus, ER, and mitochondria. Due to their sufficient membrane permeability, both probes were precisely localized to the target organelles in HeLa cells using HaloTag labeling technology. Using an in situ standard quantification method, we identified the [Zn 2+ ] in the tested organelles, resulting in the subcellular [Zn 2+ ] distribution as [

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Highlights d P5 dimerizes via a unique leucine-valine adhesive motif in the first Trx-like domain d The adhesive motif is radically different from conventional coiled-coil motifs d The dimeric structure serves to promote P5-mediated inactivation of IRE1a d Ca 2+ -binding regions and Ca 2+ -dependent functional regulation were clarified for P5

ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less-efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.