A new curcumin derivative, HBC, interferes with the cell cycle progression of colon cancer cells via antagonization of the Ca2+/calmodulin function

HBC (4-[3,5-Bis-[2-(4-hydroxy-3-methoxy-phenyl)-ethyl]-4,5-dihydro-pyrazol-1-yl]-benzoic acid) is a recently developed curcumin derivative which exhibits potent inhibitory activities against the proliferation of several tumor cell lines. In the present study, we identified Ca2+/calmodulin (Ca2+/CaM) as a direct target protein of HBC using phage display biopanning. Ca2+/CaM-expressing phages specifically bound to the immobilized HBC, and the binding was Ca2+ dependent. Moreover, flexible docking modeling demonstrated that HBC is compatible with the binding cavity for a known inhibitor, W7, in the C-terminal hydrophobic pocket of Ca2+/CaM. In biological systems, HBC induced prolonged phosphorylation of ERK1/2 and activated p21(WAF1) expression, resulting in the induction of G0/G1 cell cycle arrest in HCT15 colon cancer cells. These results suggest that HBC inhibits the cell cycle progression of colon cancer cells via antagonizing of Ca2+/CaM functions.     

The updated role of exosomal proteins in the diagnosis,prognosis, and treatment of cancer

Exosomes are vesicles encompassed by a lipid bilayer that are released by various living cells. Exosomal proteins are encapsulated within the membrane or embedded on the surface. As an important type of exosome cargo, exosomal proteins can reflect the physiological status of the parent cell and play an essential role in cell–cell communication. Exosomal proteins can regulate tumor development, including tumor-related immune regulation, microenvironment reconstruction, angiogenesis, epithelial–mesenchymal transition, metastasis, etc. The features of exosomal proteins can provide insight into exosome generation, targeting, and biological function and are potential sources of markers for cancer diagnosis, prognosis, and treatment. Here, we summarize the effects of exosomal proteins on cancer biology, the latest progress in the application of exosomal proteins in cancer diagnosis and prognosis, and the potential contribution of exosomal proteins in cancer therapeutics and vaccines.Subject terms: Protein-protein interaction networks, Cancer microenvironment     

Isolation and immunological characterization of an iron-regulated, transformation-sensitive cell surface protein of normal rat kidney cells.

We have analyzed the surfacr proteins of cultured normal rat kidney (NRK) cells and virus-transformed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation-sensitive plasma membrane-associated glycoproteins with a subunit molecular weight of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase-mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus-transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus-transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40-transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all virus-transformed cell lines. The 160 K and 130 K glycoproteins were isolated from plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane-associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.     

水分子通道蛋白的结构与功能

水分子穿越双磷脂生物膜的输运机理是生理学和细胞生物学中一个长期未能解决的重要问题.AQP1水通道蛋白的发现和鉴定使得人们确认出一个新的蛋白质家族--水通道蛋白家族.正是这一蛋白家族的存在,使得水分子可以进行快速的跨膜传输.由晶体学方法解出的哺乳动物AQP1水通道蛋白的原子结构,最终揭示了水通道蛋白只允许水分子快速传输而阻挡其他的小分子和离子(包括质子H+)的筛选输运机理.本文概述了水通道蛋白的发现和其对水分子的筛选传输机理.     

Split-luciferase complementary assay: applications,recent developments,and future perspectives

Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein–protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein–protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.     

Controlling the Substrate Specificity of an Enzyme through Structural Flexibility by Varying the Salt-Bridge Density

Many enzymes, particularly in one single family, with highly conserved structures and folds exhibit rather distinct substrate specificities. The underlying mechanism remains elusive, the resolution of which is of great importance for biochemistry, biophysics, and bioengineering. Here, we performed a neutron scattering experiment and molecular dynamics (MD) simulations on two structurally similar CYP450 proteins; CYP101 primarily catalyzes one type of ligands, then CYP2C9 can catalyze a large range of substrates. We demonstrated that it is the high density of salt bridges in CYP101 that reduces its structural flexibility, which controls the ligand access channel and the fluctuation of the catalytic pocket, thus restricting its selection on substrates. Moreover, we performed MD simulations on 146 different kinds of CYP450 proteins, spanning distinct biological categories including Fungi, Archaea, Bacteria, Protista, Animalia, and Plantae, and found the above mechanism generally valid. We demonstrated that, by fine changes of chemistry (salt-bridge density), the CYP450 superfamily can vary the structural flexibility of its member proteins among different biological categories, and thus differentiate their substrate specificities to meet the specific biological needs. As this mechanism is well-controllable and easy to be implemented, we expect it to be generally applicable in future enzymatic engineering to develop proteins of desired substrate specificities.     

Constructing real-time,wash-free,and reiterative sensors for cell surface proteins using binding-induced dynamic DNA assembly

Cell surface proteins are an important class of biomarkers for fundamental biological research and for disease diagnostics and treatment. In this communication, we report a universal strategy to construct sensors that can achieve rapid imaging of cell surface proteins without any separation by using binding-induced dynamic DNA assembly. As a proof-of-principle, we developed a real-time and wash-free sensor for an important breast cancer biomarker, human epidermal growth factor receptor-2 (HER2). We then demonstrated that this sensor could be used for imaging and sensing HER2 on both fixed and live breast cancer cells. Additionally, we have also incorporated toehold-mediated DNA strand displacement reactions into the HER2 sensor, which allows for reiterating (switching on/off) fluorescence signals for HER2 from breast cancer cells in real-time.     

Current literature highlights - June 2002

Selective Cdk4 Inhibitors: Cyclins and cyclin-dependent kinases (Cdks) play important roles in regulation of the cell cycle. In particular, D-type cyclins, which have been shown to be amplified or over-expressed in several tumour cells, associate with Cdk4/6 to activate their phosphorylation activity. Cyclin D-Cdk4/6 complexes phosphorylate the retinoblastoma protein (pRB) and regulate the cell cycle during G₁/S transition. Loss of function or deletion of p16^ink4a (endogenous Cdk4/6 specific inhibitor protein) frequently occurs in clinical cancer cells. Thus, selective Cdk4/6 inhibitors should be useful as a new class of cytostatic antitumour agents. In order to overcome the problem of selectivity for Cdk4/6 over the hundreds of homologous kinases in the superfamily, specific amino acid residues were identified around the ATP binding pocket of Cdk4 by comparing the amino acid sequences of 390 representative kinases. Subsequently, a chemical library was designed using this information about the locations of these amino acid residues (A novel approach for the development of selective Cdk4 inhibitors: Library design based on locations of Cdk4 specific amino acid residues, Honma, T. et. al., J. Med. Chem., 44, (2001), 4628-4640).     

A Novel Approach to Unraveling the Apoptotic Potential of Rutin (Bioflavonoid) via Targeting Jab1 in Cervical Cancer Cells

Rutin has been well recognized for possessing numerous pharmacological and biological activities in several human cancer cells. This research has addressed the inhibitory potential of rutin against the Jab1 oncogene in SiHa cancer cells, which is known to inactivate various tumor suppressor proteins including p53 and p27. Further, the inhibitory efficacy of rutin via Jab1 expression modulation in cervical cancer has not been yet elucidated. Hence, we hypothesized that rutin could exhibit strong inhibitory efficacy against Jab1 and, thereby, induce significant growth arrest in SiHa cancer cells in a dose-dependent manner. In our study, the cytotoxic efficacy of rutin on the proliferation of a cervical cancer cell line (SiHa) was exhibited using MTT and LDH assays. The correlation between rutin and Jab1 mRNA expression was assessed by RT-PCR analysis and the associated events (a mechanism) with this downregulation were then explored via performing ROS assay, DAPI analysis, and expression analysis of apoptosis-associated signaling molecules such as Bax, Bcl-2, and Caspase-3 and -9 using qRT-PCR analysis. Results exhibit that rutin produces anticancer effects via inducing modulation in the expression of oncogenes as well as tumor suppressor genes. Further apoptosis induction, caspase activation, and ROS generation in rutin-treated SiHa cancer cells explain the cascade of events associated with Jab1 downregulation in SiHa cancer cells. Additionally, apoptosis induction was further confirmed by the FITC-Annexin V/PI double staining method. Altogether, our research supports the feasibility of developing rutin as one of the potent drug candidates in cervical cancer management via targeting one such crucial oncogene associated with cervical cancer progression.     

Ligand specificity of CS-35, a monoclonal antibody that recognizes mycobacterial lipoarabinomannan: a model system for oligofuranoside-protein recognition

The CS-35 antibody is widely used in the characterization of glycans containing D-arabinofuranose residues, in particular polysaccharides present in the mycobacterial cell wall. A detailed understanding of the combining site of this antibody and the measurement of its binding to different ligands is of interest as this knowledge will have implications in the characterization of arabinofuranose-containing glycoconjugates that are increasingly recognized as important biological molecules. Of even greater significance is that an in-depth study of this carbohydrate-protein interaction will provide insights into the mechanisms by which oligosaccharides containing furanose rings are bound by proteins, an area that has, to date, received little attention. This system has been refractory to X-ray crystallography, and thus we report here a study of the interaction of CS-35 with its ligands using a combination of chemical synthesis, mass spectrometry, titration microcalorimetry, and NMR spectroscopy. Through these investigations we have established that the binding pocket recognizes, as a minimum epitope, a linear tetrasaccharide motif and that the residues at the reducing and non-reducing end of the oligosaccharide are essential for tight binding. The residue at the non-reducing end appears to be bound in an aliphatic pocket, whereas the rest of the tetrasaccharide interacts more strongly with aromatic amino acids.     

The Mechanism of p53 Rescue by SUSP4

p53 is an important tumor‐suppressor protein deactivation of which by mdm2 results in cancers. A SUMO‐specific protease 4 (SUSP4) was shown to rescue p53 from mdm2‐mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a “p53 rescue motif” in SUSP4 that disrupts p53‐mdm2 binding is presented. This 29‐residue motif is pre‐populated with two transient helices connected by a hydrophobic linker. The helix at the C‐terminus binds to the well‐known p53‐binding pocket in mdm2 whereas the N‐terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular‐structured SUMO‐binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti‐tumor activity in cancer cell lines expressing wild‐type p53. A pre‐structures motif in the intrinsically disordered proteins is thus important for target recognition.     

Role of Bcl-2 Family Proteins in Photodynamic Therapy Mediated Cell Survival and Regulation

Photodynamic therapy (PDT) is a treatment modality that involves three components: combination of a photosensitizer, light and molecular oxygen that leads to localized formation of reactive oxygen species (ROS). The ROS generated from this promising therapeutic modality can be lethal to the cell and leads to consequential destruction of tumor cells. However, sometimes the ROS trigger a stress response survival mechanism that helps the cells to cope with PDT-induced damage, resulting in resistance to the treatment. One preferred mechanism of cell death induced by PDT is apoptosis, and B-cell lymphoma 2 (Bcl-2) family proteins have been described as a major determinant of life or death decision of the death pathways. Apoptosis is a cellular self-destruction mechanism to remove old cells through the biological event of tissue homeostasis. The Bcl-2 family proteins act as a critical mediator of a life–death decision of cells in maintaining tissue homeostasis. There are several reports that show cancer cells developing resistance due to the increased interaction of the pro-survival Bcl-2 family proteins. However, the key mechanisms leading to apoptosis evasion and drug resistance have not been adequately understood. Therefore, it is critical to understand the mechanisms of PDT resistance, as well as the Bcl-2 family proteins, to give more insight into the treatment outcomes. In this review, we describe the role of Bcl-2 gene family proteins’ interaction in response to disease progression and PDT-induced resistance mechanisms.     

Acute response of human skin to solar radiation: regulation and function of the p53 protein.

p53 is a tumor suppressor gene and mutation of p53 is a frequent event in skin cancer. The wild-type p53 encodes for a 53-kD phosphoprotein that plays a pivotal role in regulating cell growth and cell death. The wt-p53 gene is also called "guardian of the genome", for its role in preventing the accumulation of genetic alterations, observed in cancer cells. The wild-type p53 protein plays a central role in the response of the cell to DNA damage. UV, present in sunlight, is one of the most ubiquitously present DNA damage inducing stress conditions to which skin cells are exposed. The wt-p53 protein accumulates in human skin cells in vitro and in human skin in vivo upon UV irradiation. This upregulation mounts a protective response against permanent DNA damage through transactivation of either cell cycle arrest genes and DNA repair genes or genes that mediate the apoptotic response. The molecular events which regulate the activity of the wt-p53 protein activity are only beginning to be described.     

Mutation and expression of the p27KIP1 and p57KIP2 genes in human gastric cancer

Cyclin-dependent kinase inhibitors (CDKI) are negative regulators of cell cycle progression by binding the cyclin-CDK complex and inhibiting the CDK activity. Genetic alteration in the CDKI genes has been implicated for carcinogenesis. To test the genetic alteration in the p27 and p57 genes, KIP family CDKI genes, 30 gastric tumor-normal pairs and 8 gastric cancer cell lines were analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). No mutation was detected in these genes although length polymorphisms in the proline-alanine repeat of the p57 gene were detected. When the p27 and p57 mRNAs were analyzed in gastric cancer cell lines by RT-PCR, the p27 mRNA was expressed considerably high in tumor cells but expression of the p57 mRNA was much low in gastric cancer cell lines compared to that of normal cells. The result suggests that inactivation of gene expression rather than mutations in the p57 gene accounts possibly for the involvement of this gene in tumorigenesis of gastric cancer. However, expression of the p27 gene seems to be essential for cell survival.     

Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 μM belinostat were analysed by 2‐D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC‐MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto‐oncogene proteins p53, Myc, activator protein 1, and c‐fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.     

p41-Arc, a regulatory subunit of Arp2/3 complex, can induce premature senescence in the absence of p53 and Rb

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the tumor suppressor proteins, such as Rb or p53. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-β-galactosidase (SA-β-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. p41-Arc has been known to be a putative regulatory component of the mammalian Arp2/3 complex, which is required for the formation of branched networks of actin filaments at the cell cortex. In this study, we demonstrate that p41-Arc can induce senescent phenotypes when it is overexpressed in human tumor cell line, SaOs-2, which is deficient in p53 and Rb tumor suppressor genes, implying that p41 can induce senescence in a p53-independent way. p41-Arc overexpression causes a change in actin filaments, accumulating actin filaments in nuclei. Therefore, these results imply that a change in actin filament can trigger an intrinsic senescence program in the absence of p53 and Rb tumor suppressor genes.     

A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells

BACKGROUND: The Hsp90s contain a conserved pocket that binds ATP/ADP and plays an important role in the regulation of chaperone function. Occupancy of this pocket by several natural products (geldanamycin (GM) and radicicol) alters Hsp90 function and results in the degradation of a subset of proteins (i.e. steroid receptors, Her2, Raf). We have used the structural features of this pocket to design a small molecule inhibitor of Hsp90. RESULTS: The designed small molecule PU3 competes with GM for Hsp90 binding with a relative affinity of 15-20 microM. PU3 induces degradation of proteins, including Her2, in a manner similar to GM. Furthermore, PU3 inhibits the growth of breast cancer cells causing retinoblastoma protein hypophosphorylation, G1 arrest and differentiation. CONCLUSIONS: PU3 is representative of a novel class of synthetic compounds that binds to Hsp90 and inhibits the proliferation of cancer cells. These reagents could provide a new strategy for the treatment of cancers.     

Isolation and structure elucidation of Chlorofusin, a novel p53-MDM2 antagonist from a Fusarium sp.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.     

p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O₂^•-), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O₂^•- production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b₅₅₈) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.