Molecular Mechanism and Agricultural Application of the NifA-NifL System for Nitrogen Fixation.

Nitrogen-fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N2) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the "nitrogen problem". Azotobacter vinelandii is a well-established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA-NifL system fine-tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen-fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen-fixing regulation.

Structure of an endogenous yeast 26S proteasome reveals two major conformational states.

The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Šresolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.

Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27-linked Ubiquitin Chains.

New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography. The results revealed that free K27 diUb adopted a compact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding, an interesting unique structural feature that may explain the distinctive biological function of K27 Ub chains.

Structure and mechanism of the hexameric MecA-ClpC molecular machine.

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. Bacterial ClpC, a member of the Clp/Hsp100 family of AAA+ proteins (ATPases associated with diverse cellular activities) with two nucleotide-binding domains (D1 and D2), requires the adaptor protein MecA for activation and substrate targeting. The activated, hexameric MecA-ClpC molecular machine harnesses the energy of ATP binding and hydrolysis to unfold specific substrate proteins and translocate the unfolded polypeptide to the ClpP protease for degradation. Here we report three related crystal structures: a heterodimer between MecA and the amino domain of ClpC, a heterododecamer between MecA and D2-deleted ClpC, and a hexameric complex between MecA and full-length ClpC. In conjunction with biochemical analyses, these structures reveal the organizational principles behind the hexameric MecA-ClpC complex, explain the molecular mechanisms for MecA-mediated ClpC activation and provide mechanistic insights into the function of the MecA-ClpC molecular machine. These findings have implications for related Clp/Hsp100 molecular machines.

Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer.

Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.

Structural dynamics of the MecA-ClpC complex: a type II AAA+ protein unfolding machine.

The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA(+) proteins have long been conceived, the detailed mechanistic model remains elusive. With cryoelectron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA(+) rings and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of ways, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two nucleotide-binding domain rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA(+) rings and suggest that concerted actions of the two AAA(+) rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase and especially lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA(+) hexamers.

MoS2/NiSe2/rGO Multiple-Interfaced Sandwich-like Nanostructures as Efficient Electrocatalysts for Overall Water Splitting.

Constructing a heterogeneous interface using different components is one of the effective measures to achieve the bifunctionality of nanocatalysts, while synergistic interactions between multiple interfaces can further optimize the performance of single-interface nanocatalysts. The non-precious metal nanocatalysts MoS2/NiSe2/reduced graphene oxide (rGO) bilayer sandwich-like nanostructure with multiple well-defined interfaces is prepared by a simple hydrothermal method. MoS2 and rGO are layered nanostructures with clear boundaries, and the NiSe2 nanoparticles with uniform size are sandwiched between both layered nanostructures. This multiple-interfaced sandwich-like nanostructure is prominent in catalytic water splitting with low overpotential for oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) and almost no degradation in performance after a 20 h long-term reaction. In order to simulate the actual overall water splitting process, the prepared nanostructures are assembled into MoS2/NiSe2/rGO||MoS2/NiSe2/rGO modified two-electrode system, whose overpotential is only 1.52 mV, even exceeded that of noble metal nanocatalyst (Pt/C||RuO2~1.63 mV). This work provides a feasible idea for constructing multi-interface bifunctional electrocatalysts using nanoparticle-doped bilayer-like nanostructures.

Ferroptosis: Redox Imbalance and Hematological Tumorigenesis.

Ferroptosis is a novel characterized form of cell death featured with iron-dependent lipid peroxidation, which is distinct from any known programmed cell death in the biological processes and morphological characteristics. Recent evidence points out that ferroptosis is correlated with numerous metabolic pathways, including iron homeostasis, lipid metabolism, and redox homeostasis, associating with the occurrence and treatment of hematological malignancies, such as multiple myeloma, leukemia, and lymphoma. Nowadays, utilizing ferroptosis as the target to prevent and treat hematological malignancies has become an active and challenging topic of research, and the regulatory network and physiological function of ferroptosis also need to be further elucidated. This review will summarize the recent progress in the molecular regulation of ferroptosis and the physiological roles and therapeutic potential of ferroptosis as the target in hematological malignancies.

How Effective are Nano-Based Dressings in Diabetic Wound Healing? A Comprehensive Review of Literature.

Chronic wound caused by diabetes is an important cause of disability and seriously affects the quality of life of patients. Therefore, it is of great clinical significance to develop a wound dressing that can accelerate the healing of diabetic wounds. Nanoparticles have great advantages in promoting diabetic wound healing due to their antibacterial properties, low cytotoxicity, good biocompatibility and drug delivery ability. Adding nanoparticles to the dressing matrix and using nanoparticles to deliver drugs and cytokines to promote wound healing has proven to be effective. This review will focus on the effects of diabetes on wound healing, introduce the properties, preparation methods and action mechanism of nanoparticles in wound healing, and describe the effects and application status of various nanoparticle-loaded dressings in diabetes-related chronic wound healing.

USP13: Multiple Functions and Target Inhibition.

As a deubiquitination (DUB) enzyme, ubiquitin-specific protease 13 (USP13) is involved in a myriad of cellular processes, such as mitochondrial energy metabolism, autophagy, DNA damage response, and endoplasmic reticulum-associated degradation (ERAD), by regulating the deubiquitination of diverse key substrate proteins. Thus, dysregulation of USP13 can give rise to the occurrence and development of plenty of diseases, in particular malignant tumors. Given its implications in the stabilization of disease-related proteins and oncology targets, considerable efforts have been committed to the discovery of inhibitors targeting USP13. Here, we summarize an overview of the recent advances of the structure, function of USP13, and its relations to diseases, as well as discovery and development of inhibitors, aiming to provide the theoretical basis for investigation of the molecular mechanism of USP13 action and further development of more potent druggable inhibitors.

Met1-specific motifs conserved in OTUB subfamily of green plants enable rice OTUB1 to hydrolyse Met1 ubiquitin chains.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.

Molecular determinants of MecA as a degradation tag for the ClpCP protease.

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. In Bacillus subtilis, the degradation of the competence transcription factor ComK is mediated by a ternary complex involving the adaptor protein MecA and the ATP-dependent protease ClpCP. Here we demonstrate that a C-terminal, 98-amino acid domain of MecA (residues 121-218) serves as a non-recycling, degradation tag and targets a variety of fusion proteins to the ClpCP protease for degradation. MecA-(121-218) facilitates productive oligomerization of ClpC, stimulates the ATPase activity of ClpC, and allows the activated ClpC complex to stably associate with ClpP. Importantly, the ClpCP protease undergoes dynamic cycles of assembly and disassembly, which are triggered by association with MecA and the degradation of MecA, respectively.

Crystal structure of the MecA degradation tag.

MecA is an adaptor protein that regulates the assembly and activity of the ATP-dependent ClpCP protease in Bacillus subtilis. MecA contains two domains. Although the amino-terminal domain of MecA recruits substrate proteins such as ComK and ComS, the carboxyl-terminal domain (residues 121-218) has dual roles in the regulation and function of ClpCP protease. MecA-(121-218) facilitates the assembly of ClpCP oligomer, which is required for the protease activity of ClpCP. This domain was identified to be a non-recycling degradation tag that targets heterologous fusion proteins to the ClpCP protease for degradation. To elucidate the mechanism of MecA, we determined the crystal structure of MecA-(121-218) at 2.2 A resolution, which reveals a previously uncharacterized alpha/beta fold. Structure-guided mutagenesis allows identification of surface residues that are essential for the function of MecA. We also solved the structure of a carboxyl-terminal domain of YpbH, a paralogue of MecA in B. subtilis, at 2.4 A resolution. Despite low sequence identity, the two structures share essentially the same fold. The presence of MecA homologues in other bacterial species suggests conservation of a large family of unique degradation tags.

Small molecule inhibitors reveal allosteric regulation of USP14 via steric blockade.

The ubiquitin system is important for drug discovery, and the discovery of selective small-molecule inhibitors of deubiquitinating enzymes (DUBs) remains an active yet extremely challenging task. With a few exceptions, previously developed inhibitors have been found to bind the evolutionarily conserved catalytic centers of DUBs, resulting in poor selectivity. The small molecule IU1 was the first-ever specific inhibitor identified and exhibited surprisingly excellent selectivity for USP14 over other DUBs. However, the molecular mechanism for this selectivity was elusive. Herein, we report the high-resolution co-crystal structures of the catalytic domain of USP14 bound to IU1 and three IU1 derivatives. All the structures of these complexes indicate that IU1 and its analogs bind to a previously unknown steric binding site in USP14, thus blocking the access of the C-terminus of ubiquitin to the active site of USP14 and abrogating USP14 activity. Importantly, this steric site in USP14 is very unique, as suggested by structural alignments of USP14 with several known DUB X-ray structures. These results, in conjunction with biochemical characterization, indicate a coherent steric blockade mechanism for USP14 inhibition by compounds of the IU series. In light of the recent report of steric blockade of USP7 by FT671, this work suggests a potential generally applicable allosteric mechanism for the regulation of DUBs via steric blockade, as showcased by our discovery of IU1-248 which is 10-fold more potent than IU1.

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states.

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.