The Png1–Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways

Multiple Interactions of Rad23 Suggest a Mechanism for Ubiquitylated Substrate Delivery Important in Proteolysis

The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome

Idle Vehicle Relocation Strategy through Deep Learning for Shared Autonomous Electric Vehicle System Optimization

In optimization of a shared autonomous electric vehicle (SAEV) system, idle vehicle relocation strategies are important to reduce operation costs and customers' wait time. However, for an on-demand service, continuous optimization for idle vehicle relocation is computationally expensive, and thus, not effective. This study proposes a deep learning-based algorithm that can instantly predict the optimal solution to idle vehicle relocation problems under various traffic conditions. The proposed relocation process comprises three steps. First, a deep learning-based passenger demand prediction model using taxi big data is built. Second, idle vehicle relocation problems are solved based on predicted demands, and optimal solution data are collected. Finally, a deep learning model using the optimal solution data is built to estimate the optimal strategy without solving relocation. In addition, the proposed idle vehicle relocation model is validated by applying it to optimize the SAEV system. We present an optimal service system including the design of SAEV vehicles and charging stations. Further, we demonstrate that the proposed strategy can drastically reduce operation costs and wait times for on-demand services

Usa1 protein facilitates substrate ubiquitylation through two separate domains.

BACKGROUND:Defects in protein folding are recognized as the root of many neurodegenerative disorders. In the endoplasmic reticulum (ER), secretory proteins are subjected to a stringent quality control process to eliminate misfolded proteins by the ER-associated degradation (ERAD) pathway. A novel ERAD component Usa1 was recently identified. However, the specific role of Usa1 in ERAD remains obscure. METHODOLOGY/PRINCIPAL FINDINGS:Here, we demonstrate that Usa1 is important for substrate ubiquitylation. Furthermore, we defined key cis-elements of Usa1 essential for its degradation function. Interestingly, a putative proteasome-binding motif is dispensable for the functioning of Usa1 in ERAD. We identify two separate cytosolic domains critical for Usa1 activity in ERAD, one of which is involved in binding to the Ub-protein ligase Hrd1/Hrd3. Usa1 may have another novel role in substrate ubiquitylation that is separate from the Hrd1 association. CONCLUSIONS/SIGNIFICANCE:We conclude that Usa1 has two important roles in ERAD substrate ubiquitylation

Force Measurement for the Interaction between Cucurbit[7]uril and Mica and Self-Assembled Monolayer in the Presence of Zn2+ Studied with Atomic Force Microscopy

Force spectroscopy with atomic force microscopy (AFM) revealed that cucurbit[7]uril (CB[7]) strongly binds to a mica surface in the presence of cations. Indeed, Zn2+ was observed to facilitate the self-assembly of CB [7] on the mica surface, whereas monocations, such as Nat, were less effective. The progression of the process and the cation mediated self -assembled monolayer were characterized using AFM, and the observed height of the layer agrees well with the calculated CB[7] value (9.1 A). We utilized force-based AFM to further study the interaction of CB[7] with guest molecules. To this end, CB [7] was immobilized on a glass substrate, and aminomethylferrocene (am-Fc) was conjugated onto an AFM tip. The single -molecule interaction between CB[7] and am-Fc was monitored by collecting the unbinding force curves. The force histogram showed single ruptures and a unimodal distribution, and the most probable unbinding force value was 101 pN in deionized water and 86 pN in phosphate -buffered saline buffer. The results indicate that the unbinding force was larger than that of streptavidin-biotin measured under the same conditions, whereas the dissociation constant was smaller by 1 order of magnitude (0.012 s(-1) vs 0.13 s(-1)). Furthermore, a high resolution adhesion force map showed a part of the CB[7] cavities on the surface.11sciescopu

One-pot Synthesis of a Truncated Cone-shaped Porphyrin Macrocycle and Its Self-assembly into Permanent Porous Material

© 2021 Wiley-VCH GmbHHere, we report the synthesis of a truncated cone-shaped triangular porphyrinic macrocycle, P3L3, via a single step imine condensation of a cis-diaminophenylporphyrin and a bent dialdehyde-based linker as building units. X-ray diffraction analysis reveals that the truncated cone-shaped P3L3 molecules are stacked on top of each other by π⋯π and CH⋯π interactions, to form 1.7 nm wide hollow columns in the solid state. The formation of the triangular macrocycle is corroborated by quantum chemical calculations. The permanent porosity of the P3L3 crystals is demonstrated by several gas sorption experiments and powder X-ray diffraction analysis.11Nsciescopu

Two N-terminal fragments of Usa1 are important for ERAD.

<p>(A) Domain structure of wild-type Usa1 and various deletion mutants constructed. Two short transmembrane domains (amino acids 537–583) anchor Usa1 to the ER membrane, but most of Usa1 sequences are in the cytosol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone.0007604-Carvalho1" target="_blank">[10]</a>. Grey box represents the UBL domain and black boxes indicate two transmembrane domains of Usa1. The deleted portions are shown as solid lines. The deleted region in d2Δ is the same as UBLΔ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone-0007604-g002" target="_blank">Figure 2</a>. Deletion strains and plasmids of Usa1 were constructed as described in Experimental procedures. (B) Deletions of two N-terminal segments in Usa1 impair the degradation of CPY*. Pulse chase analysis of CPY* in wild-type and various <i>USA1</i> mutant strains was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone-0007604-g002" target="_blank">Figure 2C</a>. The amount of CPY* left (%) is indicated under each lane. (C) Compromised degradation of glycosylated RTA in <i>USA1</i> deletion mutants. Pulse chase analysis of RTA was done as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone.0007604-Kim2" target="_blank">[17]</a>. (D) Usa1 deletion mutants maintain the ER membrane localization. Yeast cells expressing Flag-tagged Usa1 derivatives were labeled with ³⁵S. Protein extracts were separated into total, membrane and soluble fractions and subsequently immunoprecipitated with Flag-beads. Rad23 was used as a positive control for soluble fraction. (E) Pulse chase analysis of Usa1 wild type and its derivatives. The stabilities of Flag-tagged Usa1 alleles were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone-0007604-g002" target="_blank">Figure 2C</a>.</p

The UBL domain of Usa1 is not critical for ERAD.

<p>(A) Usa1 fails to interact with the proteasome. The plasmid expressing Flag-tagged Usa1 or Rad23 was transformed into wild type cells. To detect the association with the proteasome, the immunoprecipitates were resolved by SDS-PAGE and probed with an antibody against Rpt5, a proteasome subunit (top panel). The amounts of Rpt5, Usa1 or Rad23 in extracts are shown in lower panels. (B) Usa1 does not bind Ufd2. Proteins were extracted from cells expressing myc-tagged Ufd2 and Flag-Usa1 or Flag-Rad23. The indicated immunoprecipitation and immunoblotting were carried out as described in (A). (C) UBL domain of Usa1 is not essential for CPY* degradation. Pulse chase analysis of Flag-tagged CPY* was carried out in isogenic yeast strains BY4741 (wild-type), <i>usa1Δ</i>, YHR157 (<i>USA1</i>^ΔUBL). Yeast cells were pulse labeled with ³⁵S for 10 min, then cold Met/Cys mix was added to start the chase. We took samples at 20 min intervals and processed them for immunoprecipitation with Flag-beads, followed by SDS-PAGE and autoradiography. (D) Quantitation of the data in C. The amount of proteins was determined by phosphor-imager analysis.</p

Usa1 is required for efficient substrate ubiquitylation.

<p>(A) Usa1 is involved in CPY* ubiquitylation. CPY* was immunoprecipitated from wild-type or <i>usa1Δ</i>, <i>hrd1Δ</i> cells expressing HA-tagged Ub and analyzed by immunoblotting with anti-HA antibody. The proteasome inhibitor MG132 was added to trap CPY*. Ubiquitylated CPY* are shown on the upper panel. The bottom panel shows the levels of CPY* in these cells. Molecular weight (kDa) is indicated on the left. (B) Co-immunoprecipitation analysis of interactions between Hrd3 and Usa1. The plasmid expressing Flag-tagged Usa1 was transformed into wild type or <i>hrd1Δ</i> strains expressing Hrd3 tagged with HA epitope at its genomic locus. Proteins were extracted from the indicated cells and immunoprecipitated with Flag-beads. Immunoprecipitates were separated on SDS-PAGE, and later probed with anti-HA antibody (top panel). The amounts of Usa1 and Hrd3 in cell extracts were determined and shown in lower panels. (C) Usa1 binds Hrd1 in the absence of Hrd3. Co-immunoprecipitation analysis of interactions between Usa1 and HA-tagged Hrd1 in wild-type or <i>hrd3Δ</i> cells was conducted as described in (B). Note that Hrd1 expression is reduced in <i>hrd3Δ</i> strains as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone.0007604-Gardner1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007604#pone.0007604-Plemper1" target="_blank">[15]</a>.</p