Degradation of Polo-like Kinase 1 by the Novel Poly-Arginine N-Degron Pathway PROTAC Regulates Tumor Growth in Nonsmall Cell Lung Cancer.

Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.

Mechanically derived short-range order and its impact on the multi-principal-element alloys.

Chemical short-range order in disordered solid solutions often emerges with specific heat treatments. Unlike thermally activated ordering, mechanically derived short-range order (MSRO) in a multi-principal-element Fe40Mn40Cr10Co10 (at%) alloy originates from tensile deformation at 77 K, and its degree/extent can be tailored by adjusting the loading rates under quasistatic conditions. The mechanical response and multi-length-scale characterisation pointed to the minor contribution of MSRO formation to yield strength, mechanical twinning, and deformation-induced displacive transformation. Scanning and high-resolution transmission electron microscopy and the anlaysis of electron diffraction patterns revealed the microstructural features responsible for MSRO and the dependence of the ordering degree/extent on the applied strain rates. Here, we show that underpinned by molecular dynamics, MSRO in the alloys with low stacking-fault energies forms when loaded at 77 K, and these systems that offer different perspectives on the process of strain-induced ordering transition are driven by crystalline lattice defects (dislocations and stacking faults).

Quantitative analysis of retained austenite in Nb added Fe-based alloy.

The use of Pipelines for long-distance transportation of crude oil, natural gas and similar applications is increasing and has pivotal importance in recent times. High specific strength plays a crucial role in improving transport efficiency through increased pressure and improved laying efficiency through reduced diameter and weight of line pipes. TRIP-based high-strength and high-ductility alloys comprise a mixture of ferrite, bainite, and retained austenite that provide excellent mechanical properties such as dimensional stability, fatigue strength, and impact toughness. This study performs microstructure analysis using both Nital etching and LePera etching methods. At the time of Nital etching, it is difficult to distinctly observe second phase. However, using LePera etching conditions it is possible to distinctly measure the M/A phase and ferrite matrix. The fraction measurement was done using OM and SEM images which give similar results for the average volume fraction of the phases. Although it is possible to distinguish the M/A phase from the SEM image of the sample subjected to LePera etching. However, using Nital etching is nearly impossible. Nital etching is good at specific phase analysis than LePera etching when using SEM images.

Signaling Pathways Regulated by UBR Box-Containing E3 Ligases.

UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases.

R-catcher, a potent molecular tool to unveil the arginylome.

Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.

Synergetic strengthening of layered steel sheet investigated using an in situ neutron diffraction tensile test.

Synergetic strengthening induced by plastic strain incompatibility at the interface, and the resulting extra geometrically necessary dislocations (GNDs) generated during plastic deformation, were investigated to understand the origin of extra strength in heterogeneous structured (HS) materials. The mechanism of extra GND generation in twinning-induced plasticity (TWIP)-interstitial free (IF) steel layered sheet was quantitatively analyzed by conducting in situ neutron scattering tensile test. Load partitioning due to the different mechanical properties between the TWIP-steel core and IF-steel sheath at the TWIP/IF interface was observed during the in situ tensile testing. Because of the plastic strain incompatibility from load partitioning, extra GNDs are generated and saturate during tensile deformation. The extra GNDs can be correlated with the back-stress evolution of the HS materials, which contributes to the strength of layered materials. Because of the back-stress evolution caused by load partitioning, the strength of TWIP-IF layered steel is higher than the strength estimated by the rule-of-mixtures. This finding offers a mechanism by which extra GNDs are generated during load partitioning and shows how they contribute to the mechanical properties of HS materials.

Superior Strength and Multiple Strengthening Mechanisms in Nanocrystalline TWIP Steel.

The strengthening mechanism of the metallic material is related to the hindrance of the dislocation motion, and it is possible to achieve superior strength by maximizing these obstacles. In this study, the multiple strengthening mechanism-based nanostructured steel with high density of defects was fabricated using high-pressure torsion at room and elevated temperatures. By combining multiple strengthening mechanisms, we enhanced the strength of Fe-15 Mn-0.6C-1.5 Al steel to 2.6 GPa. We have found that solute segregation at grain boundaries achieves nanograined and nanotwinned structures with higher strength than the segregation-free counterparts. The importance of the use of multiple deformation mechanism suggests the development of a wide range of strong nanotwinned and nanostructured materials via severe plastic deformation process.

Effect of Annealing on Microstructure and Tensile Behavior of CoCrNi Medium Entropy Alloy Processed by High-Pressure Torsion.

Annealing of severely plastic deformed materials is expected to produce a good combination of strength and ductility, which has been widely demonstrated in conventional materials. In the present study, high-pressure torsion processed CoCrNi medium entropy alloy consisting of a single face-centered cubic (FCC) phase with a grain size of ~50 nm was subjected to different annealing conditions, and its effect on microstructure and mechanical behavior was investigated. The annealing of high-pressure torsion processed CoCrNi alloy exhibits partial recrystallization and near full recrystallization based on the annealing temperature and time. The samples annealed at 700 °C for 2 min exhibit very fine grain size, a high fraction of low angle grain boundaries, and high kernel average misorientation value, indicating partially recrystallized microstructure. The samples annealed for a longer duration (>2 min) exhibit relatively larger grain size, a low fraction of low angle grain boundaries, and low kernel average misorientation value, indicating nearly full recrystallized microstructure. The annealed samples with different microstructures significantly influence the uniform elongation, tensile strength, and work hardening rate. The sample annealed at 700 °C for 15 min exhibits a remarkable combination of tensile strength (~1090 MPa) and strain to failure (~41%).

Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway.

In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.

p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis.

Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.Soluble misfolded proteins that fail to be degraded by the ubiquitin proteasome system (UPS) are redirected to autophagy via specific adaptors, such as p62. Here the authors show that p62 recognises N-degrons in these proteins, acting as a N-recognin from the proteolytic N-end rule pathway, and targets these cargos to autophagosomal degradation.

Multiscale architectured materials with composition and grain size gradients manufactured using high-pressure torsion.

The concept of multiscale architectured materials is established using composition and grain size gradients. Composition-gradient nanostructured materials are produced from coarse grained interstitial free steels via carburization and high-pressure torsion. Quantitative analyses of the dislocation density using X-ray diffraction and microstructural studies clearly demonstrate the gradients of the dislocation density and grain size. The mechanical properties of the gradient materials are compared with homogeneous nanostructured carbon steel without a composition gradient in an effort to investigate the gradient effect. Based on the above observations, the potential of multiscale architecturing to open a new material property is discussed.

Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP.

The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.

Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding.

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.