Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain.

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle-dependent regulation of nuclear morphology at the molecular level.

Structural basis for the interaction between unfarnesylated progerin and the Ig-like domain of lamin A/C in premature aging disorders.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by C-terminally truncated lamin A, termed as the pre-progerin product. Progerin is a C-terminally farnesylated protein derived from pre-progerin, which causes nuclear deformation at the inner-nuclear membrane. As an alternative or additional mechanism, a farnesylation-independent abnormal interaction between the C-terminus of progerin and Ig-like domain has been proposed. However, the molecular mechanism underlying the role of unfarnesylated C-terminus of pre-progerin in HGPS remains largely unknown. In this study, we determined the crystal structures of C-terminal peptide of progerin and Ig-like domain of lamin A/C. Results showed that the C-terminal cysteine residue of progerin forms a disulfide bond with the only cysteine residue of the Ig-like domain. This finding suggested that unfarnesylated progerin can form a disulfide bond with the Ig-like domain in the lamin meshwork. The Alphafold2-assisted docking structure showed that disulfide bond formation was promoted by a weak interaction between the groove of Ig-like domain and the unfarnesylated C-terminal tail region of progerin. Our results provide molecular insights into the normal aging process as well as premature aging of humans.

Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator.

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.

Anti-Inflammatory and Neuroprotective Effects of Morin in an MPTP-Induced Parkinson's Disease Model.

Neurodegenerative diseases such as Parkinson's disease (PD) are known to be related to oxidative stress and neuroinflammation, and thus, modulating neuroinflammation offers a possible means of treating PD-associated pathologies. Morin (2',3,4',5,7-pentahydroxy flavone) is a flavonol with anti-oxidative and anti-inflammatory effects found in wines, herbs, and fruits. The present study was undertaken to determine whether a morin-containing diet has protective effects in an MPTP-induced mouse model of PD. Mice were fed a control or morin diet for 34 days, and then MPTP (30 mg/kg, i.p.) was administered daily for 5 days to induce a PD-like pathology. We found that dietary morin prevented MPTP-induced motor dysfunction and ameliorated dopaminergic neuronal damage in striatum (STR) and substantia nigra (SN) in our mouse model. Furthermore, MPTP-induced neuroinflammation was significantly reduced in mice fed morin. In vitro studies showed that morin effectively suppressed glial activations in primary microglia and astrocytes, and biochemical analysis and a docking simulation indicated that the anti-inflammatory effects of morin were mediated by blocking the extracellular signal-regulated kinase (ERK)-p65 pathway. These findings suggest that morin effectively inhibits glial activations and has potential use as a functional food ingredient with therapeutic potential for the treatment of PD and other neurodegenerative diseases associated with neuroinflammation.

Structure and function of the hypochlorous acid-induced flavoprotein RclA from Escherichia coli.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+ We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

Beta-strand-mediated dimeric formation of the Ig-like domains of human lamin A/C and B1.

Lamins are nuclear intermediate filament proteins that play an essential role in maintaining the nuclear structure by forming a 3-D meshwork. Lamins consist of the N-terminal unstructured head, the coiled-coil rod domain, and the C-terminal tail, which is mostly unstructured except for the Ig-like domain. To date, the Ig-like domain has been characterized as a monomeric structure. Here, we determined the crystal structures of human lamin A/C, including the Ig-like domain and its N- and C-terminal flanking sequences. Interestingly, the structures showed a homodimer formed by beta-strand interactions between the N- and C-terminal flanking sequences. This interaction also provides a molecular implication for the creation of a 3-D meshwork between the 3.5-nm-thick filaments. Furthermore, we determined the crystal structure of the corresponding region of lamin B1. The structure showed a similar dimeric assembly, also formed by beta-strand interactions, albeit the intersubunit distance was much shorter. Since the Ig-like domain contains many genetic hotspots causing lamin-related diseases in lamin A/C, our findings will help understand the detailed assembly of lamins in a 3-D meshwork structure and lamin-related diseases at the molecular level.

The hydrogen peroxide hypersensitivity of OxyR2 in Vibrio vulnificus depends on conformational constraints.

Most Gram-negative bacteria respond to excessive levels of H2O2 using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. Vibrio vulnificus has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2 and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H2O2 sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2 To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H2O2 was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H2O2 and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H2O2 sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2 and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.

Crystal structure of GSK3ß in complex with the flavonoid, morin.

GSK3ß is a key kinase that plays a role in cellular signaling pathways. In Alzheimer's disease (AD), GSK3ß has been implicated in hyperphosphorylation of tau proteins in the neuron, which is a hallmark of AD. Morin, a flavonoid that is abundant in nature, was found as an inhibitor of GSK3ß that can reduce tau pathology in vivo and in vitro. In this study, we determined the crystal structure of GSK3ß in complex with morin. The structure revealed that morin inhibits GSK3ß by binding to the ATP binding pocket. Our findings augment the potential of morin as a functional food to help prevent AD, as well as to provide structural information to develop new therapeutics based on the morin skeleton.

Crystal structure of ß-N-acetylglucosaminidase CbsA from Thermotoga neapolitana.

CbsA from the thermophilic marine bacteria Thermotoga neapolitana is a chitinolyitc enzyme that can cleave a glycosidic bond of the polymer N-acetylglucosamine at the non-reducing end. This enzyme has particularly high activity on di-N-acetylchitobiose. CbsA consists of a family of 3 glycoside hydrolase (GH3)-type catalytic domains and a unique C-terminal domain. The C-terminal domain distinguishes CbsA from other GH3-type enzymes. Sequence analyses have suggested that CbsA has the Asp-His dyad as a general acid/base with the NagZ of Bacillus subtilis and the Salmonella enterica serovar Typhimurium. Here, we determined the crystal structure of CbsA from T. neapolitana at a resolution of 2.0 Å using the Zn-SAD method, revealing a unique homodimeric assembly facilitated by the C-terminal domains in the dimer. We observed that CbsA is strongly inhibited by ZnCl2, and two zinc ions were consistently bound in the active site. Our results can explain the zinc ion's inhibition mechanism in the subfamily of GH3 enzymes, and provide information on the structural diversity and substrate specificity of this hydrolase family.

Lamin Filament Assembly Derived from the Atomic Structure of the Antiparallel Four-Helix Bundle.

The nucleoskeletal protein lamin is primarily responsible for the mechanical stability of the nucleus. The lamin assembly process requires the A11, A22, and ACN binding modes of the coiled-coil dimers. Although X-ray crystallography and chemical cross-linking analysis of lamin A/C have provided snapshots of A11 and ACN binding modes, the assembly mechanism of the entire filament remains to be explained. Here, we report a crystal structure of a coil 2 fragment, revealing the A22 interaction at the atomic resolution. The structure showed detailed structural features, indicating that two coiled-coil dimers of the coil 2 subdomain are separated and then re-organized into the antiparallel-four-helix bundle. Furthermore, our findings suggest that the ACN binding mode between coil 1a and the C-terminal part of coil 2 when the A11 tetramers are arranged by the A22 interactions. We propose a full assembly model of lamin A/C with the curvature around the linkers, reconciling the discrepancy between the in situ and in vitro observations. Our model accounts for the balanced elasticity and stiffness of the nuclear envelopes, which is essential in protecting the cellular nucleus from external pressure.

Crystal structure of progeria mutant S143F lamin A/C reveals increased hydrophobicity driving nuclear deformation.

Lamins are intermediate filaments that form a 3-D meshwork in the periphery of the nuclear envelope. The recent crystal structure of a long fragment of human lamin A/C visualized the tetrameric assembly unit of the central rod domain as a polymerization intermediate. A genetic mutation of S143F caused a phenotype characterized by both progeria and muscular dystrophy. In this study, we determined the crystal structure of the lamin A/C fragment harboring the S143F mutation. The obtained structure revealed the X-shaped interaction between the tetrameric units in the crystals, potentiated by the hydrophobic interactions of the mutated Phe143 residues. Subsequent studies indicated that the X-shaped interaction between the filaments plays a crucial role in disrupting the normal lamin meshwork. Our findings suggest the assembly mechanism of the 3-D meshwork and further provide a molecular framework for understanding the aging process by nuclear deformation.

Structural analysis of the overoxidized Cu/Zn-superoxide dismutase in ROS-induced ALS filament formation.

Eukaryotic Cu, Zn-superoxide dismutase (SOD1) is primarily responsible for cytotoxic filament formation in amyotrophic lateral sclerosis (ALS) neurons. Two cysteine residues in SOD1 form an intramolecular disulfide bond. This study aims to explore the molecular mechanism of SOD1 filament formation by cysteine overoxidation in sporadic ALS (sALS). In this study, we determined the crystal structure of the double mutant (C57D/C146D) SOD1 that mimics the overoxidation of the disulfide-forming cysteine residues. The structure revealed the open and relaxed conformation of loop IV containing the mutated Asp57. The double mutant SOD1 produced more contagious filaments than wild-type protein, promoting filament formation of the wild-type SOD1 proteins. Importantly, we further found that HOCl treatment to the wild-type SOD1 proteins facilitated their filament formation. We propose a feasible mechanism for SOD1 filament formation in ALS from the wild-type SOD1, suggesting that overoxidized SOD1 is a triggering factor of sALS. Our findings extend our understanding of other neurodegenerative disorders associated with ROS stresses at the molecular level.

Structure and Function of the Autolysin SagA in the Type IV Secretion System of Brucella abortus.

A recent genetic study with Brucella abortus revealed the secretion activator gene A (SagA) as an autolysin component creating pores in the peptidoglycan (PGN) layer for the type IV secretion system (T4SS) and peptidoglycan hydrolase inhibitor A (PhiA) as an inhibitor of SagA. In this study, we determined the crystal structures of both SagA and PhiA. Notably, the SagA structure contained a PGN fragment in a space between the N- and C-terminal domains, showing the substrate-dependent hinge motion of the domains. The purified SagA fully hydrolyzed the meso-diaminopimelic acid (DAP)-type PGN, showing a higher activity than hen egg-white lysozyme. The PhiA protein exhibiting tetrameric assembly failed to inhibit SagA activity in our experiments. Our findings provide implications for the molecular basis of the SagA-PhiA system of B. abortus. The development of inhibitors of SagA would further contribute to controlling brucellosis by attenuating the function of T4SS, the major virulence factor of Brucella.

Crystal structure of the nuclease and capping domain of SbcD from Staphylococcus aureus.

The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.

Human WRN is an intrinsic inhibitor of progerin, abnormal splicing product of lamin A.

Werner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.

Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

Progerinin, an optimized progerin-lamin A binding inhibitor, ameliorates premature senescence phenotypes of Hutchinson-Gilford progeria syndrome.

Previous work has revealed that progerin-lamin A binding inhibitor (JH4) can ameliorate pathological features of Hutchinson-Gilford progeria syndrome (HGPS) such as nuclear deformation, growth suppression in patient's cells, and very short life span in an in vivo mouse model. Despite its favorable effects, JH4 is rapidly eliminated in in vivo pharmacokinetic (PK) analysis. Thus, we improved its property through chemical modification and obtained an optimized drug candidate, Progerinin (SLC-D011). This chemical can extend the life span of LmnaG609G/G609G mouse for about 10 weeks and increase its body weight. Progerinin can also extend the life span of LmnaG609G/+ mouse for about 14 weeks via oral administration, whereas treatment with lonafarnib (farnesyl-transferase inhibitor) can only extend the life span of LmnaG609G/+ mouse for about two weeks. In addition, progerinin can induce histological and physiological improvement in LmnaG609G/+ mouse. These results indicate that progerinin is a strong drug candidate for HGPS.

Cleavage-Dependent Activation of ATP-Dependent Protease HslUV from Staphylococcus aureus.

HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.

Separation of Coiled-Coil Structures in Lamin A/C Is Required for the Elongation of the Filament.

Intermediate filaments (IFs) commonly have structural elements of a central α-helical coiled-coil domain consisting of coil 1a, coil 1b, coil 2, and their flanking linkers. Recently, the crystal structure of a long lamin A/C fragment was determined and showed detailed features of a tetrameric unit. The structure further suggested a new binding mode between tetramers, designated eA22, where a parallel overlap of coil 1a and coil 2 is the critical interaction. This study investigated the biochemical effects of genetic mutations causing human diseases, focusing on the eA22 interaction. The mutant proteins exhibited either weakened or augmented interactions between coil 1a and coil 2. The ensuing biochemical results indicated that the interaction requires the separation of the coiled-coils in the N-terminal of coil 1a and the C-terminal of coil 2, coupled with the structural transition in the central α-helical rod domain. This study provides insight into the role of coil 1a as a molecular regulator in the elongation of IF proteins.

Real-Time Measurement of the Liquid Amount in Cryo-Electron Microscopy Grids Using Laser Diffraction of Regular 2-D Holes of the Grids.

Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine highresolution protein structures using the limited resource of cryo-EM instrument access.