Animals ; Cell Respiration ; Electron Transport Complex III ; metabolism ; Humans ; Iron-Sulfur Proteins ; chemistry ; metabolism ; Mitochondria ; metabolism ; Peptide Fragments ; chemistry ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Subunits ; chemistry

By now, the digestive stability experiments provided by most authoritative organizations are in vitro tests. Evaluating the protein digestive stability with in vivo models should be more objective. The present study aimed to verify the in vivo digestibility of soybean β-conglycinin β-subunit in Wuzhishan (WZS) minipigs. Three minipigs were surgically fitted with O-stomach and T-ileum cannulae and fed with soybean meals. According to SDS-PAGE, the 50 kD fraction of soybean β-conglycinin β-subunit persisted in the gastric fluid until 6 h after feeding, which was detected at 3 h and clearly visible at 4-6 h in the intestinal fluid. Western blot with anti-β-conglycinin β-subunit McAb confirmed it.
Animals ; Antigens, Plant ; chemistry ; metabolism ; Digestion ; physiology ; Globulins ; chemistry ; metabolism ; Male ; Protein Subunits ; chemistry ; metabolism ; Seed Storage Proteins ; chemistry ; metabolism ; Soybean Proteins ; chemistry ; metabolism ; Swine ; Swine, Miniature ; physiology

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.
Crystallography, X-Ray ; GTP Phosphohydrolases ; chemistry ; metabolism ; Humans ; Protein Domains ; Ribosome Subunits, Large, Eukaryotic ; chemistry ; metabolism ; Saccharomyces cerevisiae ; chemistry ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; metabolism

Large-conductance Ca²⁺-activated K⁺ channels (BK channels) constitute an key physiological link between cellular Ca²⁺ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K⁺ channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca²⁺, transmembrane voltage, and auxiliary subunit proteins.
Animals ; Calcium Signaling ; Cytoplasm ; metabolism ; Electric Conductivity ; Electrophysiological Phenomena ; Humans ; Ion Channel Gating ; Large-Conductance Calcium-Activated Potassium Channels ; chemistry ; metabolism ; Protein Subunits ; chemistry ; metabolism

The lipid droplet (LD) is a unique multi-functional organelle that contains a neutral lipid core covered with a phospholipid monolayer membrane. The LDs have been found in almost all organisms from bacteria to humans with similar shape. Several conserved functions of LDs have been revealed by recent studies, including lipid metabolism and trafficking, as well as nucleic acid binding and protection. We summarized these findings and proposed a hypothesis that the LD is a conserved organelle.
Animals ; Bacteria ; metabolism ; ultrastructure ; Biological Evolution ; Cholesterol Esters ; metabolism ; Humans ; Lipid Droplets ; chemistry ; metabolism ; ultrastructure ; Lipid Metabolism ; genetics ; Nucleic Acids ; metabolism ; Peptide Initiation Factors ; chemistry ; metabolism ; Protein Binding ; RNA-Binding Proteins ; chemistry ; metabolism ; Ribosome Subunits ; chemistry ; metabolism ; Triglycerides ; metabolism

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry

Vimentin is an intermediate filament that regulates cell attachment and subcellular organization. In this study, vimentin filaments were morphologically altered, and its soluble subunits were rapidly reduced via cadmium chloride treatment. Cadmium chloride stimulated three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and led apoptotic pathway via caspase-9 and caspase-3 activations. In order to determine whether MAPKs were involved in this cadmium-induced soluble vimentin disappearance, we applied MAPK- specific inhibitors (PD98059, SP600125, SB203580). These inhibitors did not abolish the cadmium-induced soluble vimentin disappearance. Caspase and proteosome degradation pathway were also not involved in soluble vimentin disappearance. When we observed vimentin levels in soluble and insoluble fractions, soluble vimentin subunits shifted to an insoluble fraction. As we discovered that heat- shock protein 27 (HSP27) was colocalized and physically associated with vimentin in unstressed cells, the roles of HSP27 with regard to vimentin were assessed. HSP27-overexpressing cells prevented morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the cadmium-treated cells. Moreover, HSP27 antisense oligonucleotide augmented these cadmium-induced changes in vimentin. These findings indicate that HSP27 prevents disruption of the vimentin intermediate filament networks and soluble vimentin disappearance, by virtue of its physical interaction with vimentin in cadmium-treated SK-N-SH cells.
Cadmium/*pharmacology ; Caspases/metabolism ; Cell Line ; Heat-Shock Proteins/*metabolism ; Humans ; Mitogen-Activated Protein Kinases/metabolism ; Protein Binding/drug effects ; Protein Subunits/chemistry/metabolism ; Solubility/drug effects ; Vimentin/*chemistry/*metabolism

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.
Cloning, Molecular ; DNA Polymerase III ; chemistry ; genetics ; metabolism ; DNA Replication ; DNA, Bacterial ; biosynthesis ; genetics ; Escherichia coli ; enzymology ; genetics ; Plasmids ; metabolism ; Polymerization ; Protein Engineering ; methods ; Protein Subunits ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism

Hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1beta subunit and one of three subunits (HIF-1alpha, HIF-2alpha or HIF-3alpha). The stability and activity of HIF-1alpha are regulated by various post-translational modifications, hydroxylation, acetylation, and phosphorylation. Therefore, HIF-1alpha interacts with several protein factors including PHD, pVHL, ARD-1, and p300/CBP. Under normoxia, the HIF-1alpha subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)- mediated ubiquitin-proteasome pathway. The association of pVHL and HIF-1alpha under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, in the hypoxia condition, HIF-1alpha subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Eventually, HIF-1 acts as a master regulator of numerous hypoxia-inducible genes under hypoxic conditions. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation/survival, and glucose/iron metabolism. Moreover, it was reported that the activation of HIF-1alpha is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1a itself or HIF-1alpha interacting proteins inhibit tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. This review summarizes the molecular mechanism of HIF-1a stability, the biological functions of HIF-1 and its potential applications of cancer therapies.
Alternative Splicing ; Gene Expression Regulation ; Gene Therapy ; Growth Substances/metabolism ; Humans ; Protein Isoforms/chemistry/genetics/*metabolism ; Protein Subunits/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Transcription Factors/chemistry/genetics/*metabolism ; Transcription, Genetic

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.
Acetylation ; Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Fructosediphosphates ; chemistry ; metabolism ; Gene Expression ; Humans ; Kinetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Processing, Post-Translational ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured