Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.
Amino Acid Motifs ; Aminoacyltransferases ; drug effects ; genetics ; Antigens, Bacterial ; drug effects ; Bacterial Adhesion ; drug effects ; Bacterial Proteins ; drug effects ; genetics ; Biofilms ; drug effects ; Cysteine Endopeptidases ; drug effects ; genetics ; Dose-Response Relationship, Drug ; Humans ; Mutation ; genetics ; Nicotine ; administration & dosage ; pharmacology ; Peptidoglycan ; drug effects ; genetics ; Saliva ; physiology ; Streptococcus mutans ; drug effects ; enzymology ; growth & development ; Sucrose ; pharmacology

This study was performed to investigate the effects of different regions of the Autographa califor nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localization in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect Sf9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluorescence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain (2-45aa) of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells; and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25-EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot-like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the cells transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well; and the sequence downstream of the N-terminal domain also affects trafficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most cases, suggesting that the E25 likely exists and functions as dimmers or polymers. Production of infectious BV was dramatically reduced and even completely eliminated in most cases, either in the absence or presence of the native e25.
Amino Acid Motifs ; Animals ; Cell Nucleus ; metabolism ; virology ; Mutation ; Nucleopolyhedrovirus ; chemistry ; genetics ; physiology ; Protein Transport ; Spodoptera ; virology ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virus Release ; Virus Replication

Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson's disease (PD). We and others reported recently that expression of the pathogenic gainof-function mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fission in neurons through DLP1. Here we provide evidence that expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fission through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.
Amino Acid Substitution ; Autophagosomes ; metabolism ; pathology ; Autophagy-Related Protein-1 Homolog ; chemistry ; genetics ; metabolism ; GTP Phosphohydrolases ; antagonists & inhibitors ; genetics ; metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; chemistry ; genetics ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mitochondrial Degradation ; genetics ; physiology ; Mitochondrial Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Parkinson Disease ; genetics ; metabolism ; pathology ; Protein Interaction Domains and Motifs ; Recombinant Proteins ; chemistry ; genetics ; metabolism

The importance of NAC (named as NAM, ATAF1, 2, and CUC2) proteins in plant development, transcription regulation and regulatory pathways involving protein-protein interactions has been increasingly recognized. We report here the high resolution crystal structure of SNAC1 (stress-responsive NAC) NAC domain at 2.5 Å. Although the structure of the SNAC1 NAC domain shares a structural similarity with the reported structure of the ANAC NAC1 domain, some key features, especially relating to two loop regions which potentially take the responsibility for DNA-binding, distinguish the SNAC1 NAC domain from other reported NAC structures. Moreover, the dimerization of the SNAC1 NAC domain is demonstrated by both soluble and crystalline conditions, suggesting this dimeric state should be conserved in this type of NAC family. Additionally, we discuss the possible NAC-DNA binding model according to the structure and reported biological evidences.
Amino Acid Motifs ; Amino Acid Sequence ; Conserved Sequence ; Crystallography, X-Ray ; DNA ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Oryza ; metabolism ; physiology ; Plant Proteins ; chemistry ; metabolism ; Promoter Regions, Genetic ; genetics ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Stress, Physiological

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
Amino Acid Sequence ; Binding Sites ; Calcium ; physiology ; Calcium-Binding Proteins ; chemistry ; Crystallography, X-Ray ; EF Hand Motifs ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Tertiary ; Sequence Alignment ; Sequence Homology, Amino Acid ; Streptomyces coelicolor ; Structural Homology, Protein ; Surface Properties

OBJECTIVETo study the role of HLB-1 in regulating the organization and function of neuromuscular junctions in nematode Caenorhabditis elegans.

METHODSTo evaluate the functions of HLB-1 in regulating the organization and function of neuromuscular junctions, effects of hlb-1 mutation on the synaptic structures were revealed by uncovering the expression patterns of SNB-1::GFP and UNC-49::GFP, and pharmacologic assays with aldicarb and levamisole were also used to test the synaptic functions. Further rescue and mosaic analysis confirmed HLB-1's role in regulating the organization and function of neuromuscular junctions.

RESULTSLoss of HLB-1 function did not result in defects in neuronal outgrowth or neuronal loss, but caused obvious defects of SNB-1::GFP and UNC-49::GFP puncta localization, suggesting the altered presynaptic and postsynaptic structures. The mutant animals exhibited severe defects in locomotion behaviors and altered responses to an inhibitor of acetylcholinesterase and a cholinergic agonist, indicating the altered presynaptic and postsynaptic functions. Rescue and mosaic analysis experiments suggested that HLB-1 regulated synaptic functions in a cell nonautonomously way. Moreover, HLB-1 expression was not required for the presynaptic active zone morphology. Genetic evidence further demonstrated that hlb-1 acted in a parallel pathway with syd-2 to regulate the synaptic functions.

CONCLUSIONHLB-1 appeared as a new regulator for the organization and function of neuromuscular junctions in C. elegans.

Age Factors ; Amino Acid Motifs ; physiology ; Analysis of Variance ; Animals ; Animals, Genetically Modified ; Animals, Newborn ; Behavior, Animal ; physiology ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; physiology ; Carrier Proteins ; metabolism ; Cell Adhesion Molecules ; genetics ; physiology ; Green Fluorescent Proteins ; genetics ; Locomotion ; genetics ; Mutation ; physiology ; Neuromuscular Junction ; genetics ; physiology ; Phosphoproteins ; genetics ; physiology

KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.
Amino Acid Motifs/physiology ; Amino Acid Sequence ; Antigens, Neoplasm/chemistry/*metabolism ; Cells, Cultured ; Centrosome/*metabolism ; Fluorescent Antibody Technique ; Humans ; Leucine Zippers/*physiology ; Molecular Sequence Data ; Mutation ; Nuclear Proteins/chemistry/*metabolism ; Protein Conformation ; Protein Structure, Tertiary ; Sequence Analysis, Protein

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.
Amino Acid Motifs ; Animals ; Cell Adhesion/*drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Matrix Proteins/chemistry/genetics/pharmacology ; Fibroblasts/cytology/drug effects/physiology ; Fibronectins/chemistry/genetics/*pharmacology ; Humans ; Keratinocytes/cytology/drug effects/physiology ; Mice ; NIH 3T3 Cells ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/pharmacology ; Transforming Growth Factor beta/chemistry/genetics/pharmacology ; Wound Healing/*drug effects/physiology

The largest subunit of eukaryotic RNA polymerase II contains a unique domain at its carboxy-terminus, which is referred to as the carboxy-terminal domain (CTD). The CTD is made up of an evolutionarily conserved heptapeptide repeat (YSPTSPS). Over the past decade, there has been increasing attention on the role of the CTD in transcription regulation in the view of mRNA processing and chromatin remodeling. This paper provides a brief overview of the recent progress in the dynamic changes in CTD phosphorylation and its role in integrating multiple nuclear events.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Chromatin/*physiology ; Chromatin Assembly and Disassembly ; Conserved Sequence ; Histones/metabolism ; Humans ; Isomerism ; Phosphorylation ; Protein Structure, Tertiary ; RNA Polymerase II/chemistry/*physiology ; RNA, Messenger/metabolism ; Transcription, Genetic

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
src-Family Kinases/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/*physiology ; Signal Transduction/*physiology ; Receptors, Vitronectin/genetics/*physiology ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Paxillin/metabolism ; Myocytes, Smooth Muscle/drug effects/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects/*metabolism ; Morpholines/pharmacology ; Molecular Sequence Data ; Integrins/genetics/*physiology ; Humans ; Flavonoids/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics/*physiology ; Enzyme Inhibitors/pharmacology ; Chromones/pharmacology ; Cells, Cultured ; Cell Movement/*physiology ; Cell Adhesion/physiology ; Animals ; Amino Acid Sequence ; Amino Acid Motifs/genetics ; 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors