Cloning of BNIP3h, a member of proapoptotic BNIP3 family genes

Apoptosis is regulated by interaction of antiapoptotic Bcl-2 family proteins with various proapoptotic proteins, several of which are also members of the Bcl-2 family. BNIP3 (formerly NIP3) is a proapoptotic mitochondrial protein classified in the Bcl-2 family based on limited sequence homology-3 (BH3) domain and COOH-terminal transmembrane domain. Sequence comparison of BNIP3 has indicated that there are several BNIP3 human homologs of this protein, like BNIP3L, Nix and BNIP3. We have cloned a new member of BNIP3 family from the cDNA library prepared from human dermal papilla cells and designated as BNIP3h. BNIP3h shows substantial homology with other BNIP3 family proteins. BNIP3h induced apoptosis from 24 hours after transfection in MCF7 cell lines and its apoptosis inducing activity is extended until 72 hours after transfection.     

Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.     

Temperature inducible beta-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of beta-structure with rising temperatures in all activation domain peptides. The high stability of beta-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce alpha-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L78E79-->D78L79) showed wild-type structure, the M32 mutant peptide (L78L81L83-->A78A81A83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a beta-structural element is involved in binding to a cellular cofactor.     

The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family

The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.     

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

Background

The inorganic (P_i) phosphate transporter (PiT) family comprises known and putative Na⁺- or H⁺-dependent P_i-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with P_i to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na⁺-dependent P_i (NaP_i) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins.

Results

We show that the human PiT2 histidine, H₅₀₂, and the human PiT1 glutamate, E₇₀, - both conserved in eukaryotic PiT family members - are critical for P_i transport function. Noticeably, human PiT2 H₅₀₂ is located in the C-terminal PiT family signature sequence, and human PiT1 E₇₀ is located in ProDom domains characteristic for all PiT family members. A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR₂₅₄-V₄₈₃), was found to be a fully functional P_i transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL₁₈₃-V₄₈₃) did also support P_i transport albeit at very low levels.

Conclusions

The results suggest that the overall structure of the P_i-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.     

Deliberate manipulation of the surface hydrophobicity of an adsorbent for an efficient purification of a giant molecule with multiple subunits

Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 μmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

Proteomics analysis of in vitro protein methylation during Src-induced transformation

Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and β-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of β-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.     

Visualizing Gene Expression in Living Mammals Using a Bioluminescent Reporter

Abstract— Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.     

The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins

Small heat shock proteins (sHsp) form a large ubiquitous family of proteins expressed in all phyla of living organisms. The members of this family have low molecular masses (13-43 kDa) and contain a conservative α-crystallin domain. This domain (about 90 residues) consists of several β-strands forming two β-sheets packed in immunoglobulinlike manner. The α-crystallin domain plays an important role in formation of stable sHsp dimers, which are the building blocks of the large sHsp oligomers. A large N-terminal domain and a short C-terminal extension flank the α-crystallin domain. Both the N-terminal domain and the C-terminal extension are flexible, susceptible to proteolysis, prone to posttranslational modifications, and are predominantly intrinsically disordered. Differently oriented N-terminal domains interact with each other, with the core α-crystallin domain of the same or neighboring dimers and play important role in formation of large sHsp oligomers. Phosphorylation of certain sites in the N-terminal domain affects the sHsp quaternary structure, the sHsp interaction with target proteins and the sHsp chaperone-like activity. The C-terminal extension often carrying the conservative tripeptide (I/V/L)-X-(I/V/L) is capable of binding to a hydrophobic groove on the surface of the core α-crystallin domain of neighboring dimer, thus affecting the plasticity and the overall structure of sHsp oligomers. The Cterminal extension interacts with target proteins and affects their interaction with the α-crystallin domain increasing solubility of the complexes formed by sHsp and their targets. Thus, disordered N- and C-terminal sequences play important role in the structure, regulation and functioning of sHsp.     

Molecular variations in Th1-specific cell surface gene Tim-3

The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is identified to be expressed on T cells. A member of Tim family, Tim-3 (T cell immunoglobulin mucin 3) is selectively expressed on the surface of differentiated Th1 cells. Tim-3 might have an important role in the induction of autoimmune diseases by regulating macrophage activation and interacts with Tim-3 ligand to regulate Th1 responses. To determine the variation sites in the coding and promoter region of human Tim-3 gene, we performed variation scanning by direct sequencing using the genomic DNA isolated from the patients with asthma or allergic rhinitis and healthy controls without asthma and allergic rhinitis. We identified four single nucleotide polymorphisms (SNPs) including one novel SNPs (-1541C>T) and two variation sites (-1292_-1289delTAAA and -1282_-1278dupTAAAA) in the coding and promoter region of human Tim-3 gene in both the patients and healthy groups.     

Autoacetylation induced specific structural changes in histone acetyltransferase domain of p300: probed by surface enhanced Raman spectroscopy

Reversible acetylation of histone and non-histone proteins plays an important role in the regulation of gene expression and cellular homeostasis. A balance between acetylation and deacetylation of these proteins are maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Among different HATs, p300/CBP is the most widely studied chromatin modifying enzymes. p300 is involved in several physiological processes like cell growth, regulation of gene expression, development, and tumor suppressor, and therefore its dysfunction causes different diseases. The autoacetylation of p300 is one of the key regulators of its catalytic activity. Mechanistically, autoacetylation induced structural changes in the p300 HAT domain acts as a master switch. In this report, we have shown that the natural HAT inhibitor garcinol could potently inhibit the autoacetylation activity. Furthermore, for the first time, we demonstrate that indeed autoacetylation induces structural changes in p300 HAT domain, as probed by surface-enhanced Raman scattering. Presumably, SERS will be a very useful tool to find out the structural changes in the other self-modifying enzymes like kinases and methyltransferases.