This study was aimed to investigate the effects of blockade of Ca(2+) activated channel KCa3.1 and voltage-gated potassium channel Kv1.3 of the monocytes/macrophages on inflammatory monocyte chemotaxis. Chemotaxis assay was used to test the inflammatory Ly-6C(hi) monocyte chemotaxis caused by the monocytes/macrophages. The proliferation of monocytes/macrophages was detected by cell counting kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was applied to detect the C-C motif ligand 7 (CCL7) in cultured media. The results showed that the recruitment of Ly-6C(hi) monocyte induced by monocytes/macrophages was suppressed by the potent Kv1.3 blocker Stichodactyla helianthus neurotoxin (ShK) or the specific KCa3.1 inhibitor TRAM-34. Meanwhile, the proliferation of monocytes/macrophages was significantly inhibited by ShK. The response of Ly-6C(hi) monocyte pretreated with ShK or TRAM-34 to CCL2 was declined. These results suggest that KCa3.1 and Kv1.3 may play an important role in monocytes/macrophages' proliferation and migration.
Cell Movement ; Cell Proliferation ; Cnidarian Venoms ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Kv1.3 Potassium Channel ; antagonists & inhibitors ; physiology ; Macrophages ; cytology ; Monocytes ; cytology ; Protein Structure, Tertiary ; Pyrazoles ; pharmacology ; Small-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; physiology

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.
Crystallography, X-Ray ; Ebolavirus ; physiology ; Humans ; Nucleoproteins ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship ; Virus Assembly ; physiology

With growing accounts of inflammatory diseases such as sepsis, greater understanding the immune system and the mechanisms of cellular immunity have become primary objectives in immunology studies. High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is implicated in various aspects of the innate immune system as a damage-associated molecular pattern molecule and a late mediator of inflammation, as well as in principal cellular processes, such as autophagy and apoptosis. HMGB1 functions in the nucleus as a DNA chaperone; however, it exhibits cytokine-like activity when secreted by injurious or infectious stimuli. Extracellular HMGB1 acts through specific receptors to promote activation of the NF-kappaB signaling pathway, leading to production of cytokines and chemokines. These findings further implicate HMGB1 in lethal inflammatory diseases as a crucial regulator of inflammatory, injurious, and infectious responses. In this paper, we summarize the role of HMGB1 in inflammatory and non-inflammatory states and assess potential therapeutic approaches targeting HMGB1 in inflammatory diseases.
Amino Acid Sequence ; HMGB1 Protein/chemistry/metabolism/*physiology ; Humans ; Immunity, Innate/*physiology ; *Models, Immunological ; Molecular Sequence Data ; Protein Structure, Tertiary ; Signal Transduction

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.
Acids ; chemistry ; Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; metabolism ; physiology ; HEK293 Cells ; Host-Pathogen Interactions ; Humans ; Hydrogen-Ion Concentration ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Molecular Docking Simulation ; Molecular Sequence Data ; Protein Binding ; Protein Conformation ; Protein Interaction Mapping ; Protein Structure, Tertiary ; RNA, Viral ; genetics ; metabolism ; Receptors, Scavenger ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Sf9 Cells ; Static Electricity ; Virion ; genetics ; metabolism ; Virus Attachment

In all six members of TRPV channel subfamily, there is an ankyrin repeat domain (ARD) in their intracellular N-termini. Ankyrin (ANK) repeat, a common motif with typically 33 residues in each repeat, is primarily involved in protein-protein interactions. Despite the sequence similarity among the ARDs of TRPV channels, the structure of TRPV3-ARD, however, remains unknown. Here, we report the crystal structure of TRPV3-ARD solved at 1.95 Å resolution, which reveals six-ankyrin repeats. While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily; it, however, features a noticeable finger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic packing, instead of being flexible as seen in known TRPV-ARD structures. Electrophysiological recordings demonstrated that mutating key residues R225, R226, Q255, and F249 of finger 3 loop altered the channel activities and pharmacology. Taken all together, our findings show that TRPV3-ARD with characteristic finger 3 loop likely plays an important role in channel function and pharmacology.
Amino Acid Sequence ; Ankyrin Repeat ; Crystallography, X-Ray ; HEK293 Cells ; Humans ; Models, Molecular ; Molecular Sequence Data ; Patch-Clamp Techniques ; Protein Binding ; Protein Conformation ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid ; TRPV Cation Channels ; chemistry ; physiology

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.
Arginine ; chemistry ; Aspartic Acid ; chemistry ; Cell Membrane ; physiology ; Conserved Sequence ; Ion Channel Gating ; Ion Channels ; chemistry ; Membrane Potentials ; Protein Structure, Tertiary

The p53 tumor suppressor is a sequence-specific transcription factor that undergoes an abundance of post-translational modifications for its regulation and activation. Acetylation of p53 is an important reversible enzymatic process that occurs in response to DNA damage and genotoxic stress and is indispensible for p53 transcriptional activity. p53 was the first non-histone protein shown to be acetylated by histone acetyl transferases, and a number of more recent in vivo models have underscored the importance of this type of modification for p53 activity. Here, we review the current knowledge and recent findings of p53 acetylation and deacetylation and discuss the implications of these processes for the p53 pathway.
Acetylation ; Animals ; DNA Damage ; Gene Expression Regulation ; Histone Acetyltransferases ; metabolism ; Humans ; Mice ; Phosphorylation ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; genetics ; Signal Transduction ; physiology ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Ubiquitination

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.
Amino Acid Substitution ; Animals ; Arginine ; chemistry ; metabolism ; Cell Line ; Cercopithecus aethiops ; HIV Envelope Protein gp41 ; chemistry ; metabolism ; HIV-1 ; metabolism ; Humans ; Kinetics ; Membrane Fusion ; physiology ; Mutation ; Protein Structure, Tertiary

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

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.
Animals ; Ankyrin Repeat ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Protein Subunits ; TRPC Cation Channels ; chemistry ; genetics ; physiology