Unraveling Potential Candidate Targets Associated with Expression of p16INK4a or p16 Truncated Fragment by Comparative Proteomics Analysis

2021 ◽  
Vol 18 ◽  
Author(s):  
Najmeh Fahham ◽  
Fatemeh Zandi ◽  
Mohammad Hossein Ghahremani ◽  
Seyed Nasser Ostad ◽  
Behrouz Vaziri ◽  
...  
Keyword(s):  
Stress Protein ◽  
Full Length ◽  
Functional Domain ◽  
Potential Candidate ◽  
Protein Extract ◽  
Two Dimensional Gel Electrophoresis ◽  
New Findings ◽  
Mass Spectrometry Identification ◽  
Fibrosarcoma Cells ◽  
Cycle Regulation

Background: P16 is a tumor suppressor protein that is significantly involved in cycle regulation through the reduction of cell progression from G1 phase to S phase via CDK-cyclin D/p16INK4a/pRb/E2F cascade. The minimum functional domain of p16 has been uncovered that may function comparable to wild type p16. Objective: To expand the knowledge on molecules and mechanisms by which p16 or p1666-156 fragment suppresses human fibrosarcoma cell line growth, differential proteome profiles of fibrosarcoma cells following p16 full length or the functional domain overexpression were analyzed. Methods: Following transfecting HT-1080 fibrosarcoma cells with p16 full length, p1666-156 truncated form, and pcDNA3.1 empty vector, protein extract of each sample was harvested and clarified by centrifugation, and then the protein content was determined via Bradford assay. All protein extract of each sample was analyzed by two-dimensional gel electrophoresis. Immunoblot analysis was performed as further validation of the expression status of identified proteins. Results: Expression of p16 or p1666-156 fragment could induce mostly common alterations (up/down-regulation) of proteome profile of HT-1080 cells. Mass spectrometry identification of the differentially expressed protein spots revealed several proteins that were grouped in functional clusters, including cell cycle regulation and proliferation, cell migration and structure, oxidative stress, protein metabolism, epigenetic regulation, and signal transduction. Conclusion: The minimum functional domain of p16 could act in the same way as p16 full length. Also, these new findings can significantly enrich the understanding of p16 growth-suppressive function at the molecular level by the introduction of potential candidate targets for new treatment strategies. Furthermore, the present study provides strong evidence on the functional efficacy of the identified fragment of p16 for further attempts toward peptidomimetic drug design or gene transfer to block cancer cell proliferation.

scholarly journals Beta-centractin: characterization and distribution of a new member of the centractin family of actin-related proteins.

1994 ◽  
Vol 5 (12) ◽  
pp. 1301-1310 ◽  
Author(s):  
S W Clark ◽  
O Staub ◽  
I B Clark ◽  
E L Holzbaur ◽  
B M Paschal ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cdna Clone ◽  
Expressed Sequence Tag ◽  
Full Length ◽  
Northern Analysis ◽  
Related Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Full Length Cdna ◽  
Dynactin Complex ◽  
Expressed Sequence

An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.

PROTEIN PROFILING OF DIFFERENT PLANT TISSUES FROM HERB PHYLLANTHUS NIRURI

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Ainul Mardhiah Mohd Nail ◽  
Noor Hasniza Md Zin
Keyword(s):  
Molecular Weight ◽  
Protein Band ◽  
Protein Profiling ◽  
Protein Extract ◽  
Phyllanthus Niruri ◽  
Two Dimensional Gel Electrophoresis ◽  
Plant Parts ◽  
Sds Page ◽  
Isoelectric Points

Herb Phyllanthus niruri (P. niruri) is known to have various pharmacological functions including anticancer, antibacterial, antioxidant, anti-hypertensive and also anti-diabetic properties.  In this research, the proteomic part of P. niruri was studied to determine the bioactive peptides that responsible for specific characteristics. Total soluble proteins from different plant parts of freshly collected P. niruri were extracted using TCA/acetone method and then quantified using Bradford assay. Fruits part was found to have a significantly higher amount of proteins (4.91µg/µl + 0.21) compared to leaves (4.18µg/µl + 0.15). To determine the quality of proteins in the crude extract, SDS-Page was carried out which separates proteins in the basis of molecular weight. Proteins extracted from leaves were widely distributed between the range of 3.5 kDa to 160 kDA. Meanwhile, proteins in fruits mainly distributed within the range of 15 kDa to 80 kDa. The most highly expressed protein band was found in fruit, located in between 30 to 40 kDa. The protein extracts were then further analyzed based on the molecular weight and isoelectric points using two-dimensional gel electrophoresis (2D-GE) approach. Based on the profile pattern obtained from 2D-GE analysis, protein extract from fruits seems to express more protein spots compared to protein extract from leaves. Protein spots from fruit are seen to be intensely resolved within pH 4 to 10 at molecular weight between 10 kDa to 80 kDa. On the other hand, protein spots from leaves were moderately resolved at pH 4 to 10 at molecular weight within 10 kDa to 50 kDa.

Taxonomic and nomenclatural notes on the Rhinusa tetra (Fabricius) species complex (Coleoptera: Curculionidae)

Zootaxa ◽  
2012 ◽  
Vol 3329 (1) ◽  
pp. 31 ◽  
Author(s):  
ROBERTO CALDARA ◽  
ROBERTO CASALINI ◽  
COSIMO BAVIERA
Keyword(s):  
Biological Control ◽  
North America ◽  
Species Complex ◽  
Broad Sense ◽  
Valid Species ◽  
Potential Candidate ◽  
Verbascum Thapsus ◽  
New Findings ◽  
Common Mullein

All taxa closely related to or synonymized with Rhinusa tetra (Fabricius, 1792) are studied, including the available type mate-rial. Four species are considered taxonomically valid: Rhinusa tetra, R. comosa (Rosenschoeld, 1838), R. moroderi (Reitter,1906), R. verbasci (Rosenschoeld, 1838). The following four new synonymies are proposed: R. tetra (= Gymnetron eoumRosenschoeld, 1838 syn. n.; = Cleopus uncinatus Dufour, 1843 syn. n.; = Cleopus verbasci Dufour, 1843 syn. n.); R. moroderi(= Gymnetron otini Hustache, 1946 syn. n). Neotypes are designated for Cionus amictus Germar, 1821, Cleopus uncinatus andCleopus verbasci. Lectotypes are designated for Curculio teter, Gymnetron comosum, Gymnetron crassirostre Lucas, 1849,Gymnetron eoum, Gymnetron fuscescens Rosenschoeld, 1838, Gymnetron haemorrhoum Rosenhauer, 1847, Gymnetron moro-deri, Gymnetron plagiellum Gyllenhal, 1838, Gymnetron trigonale Gyllenhal, 1838 and Gymnetron verbasci, all currentlyincluded in Rhinusa. A key separating the four valid species is supported by diagnoses, biological notes, distributional data andillustrations. These new findings are important because R. tetra in the broad sense was proposed as a potential candidate for the biological control of invasive Common Mullein (Verbascum thapsus L.) in North America.

scholarly journals Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments

1998 ◽  
Vol 18 (6) ◽  
pp. 3540-3551 ◽  
Author(s):  
Susan F. Law ◽  
Yu-Zhu Zhang ◽  
Andres J. P. Klein-Szanto ◽  
Erica A. Golemis
Keyword(s):  
Cell Cycle ◽  
Regulatory Protein ◽  
Focal Adhesions ◽  
Lymphoid Cells ◽  
Full Length ◽  
Amino Terminal ◽  
Multiple Protein ◽  
Docking Proteins ◽  
Carboxy Terminal ◽  
Cycle Regulation

ABSTRACT HEF1, p130Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115HEF1, p105HEF1, p65HEF1, and p55HEF1. We show that p115HEF1 and p105HEF1 are different phosphorylation states of the full-length HEF1. p55HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105HEF1 and p115HEF1 being rapidly upregulated upon induction of cell growth, whereas p55HEF1 is produced specifically at mitosis. While p105HEF1 and p115HEF1 are predominantly cytoplasmic and localize to focal adhesions, p55HEF1 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.

Molecular basis of albinism in India: Evaluation of seven potential candidate genes and some new findings

Gene ◽  
2012 ◽  
Vol 511 (2) ◽  
pp. 470-474 ◽  
Author(s):  
M. Mondal ◽  
M. Sengupta ◽  
S. Samanta ◽  
A. Sil ◽  
K. Ray
Keyword(s):  
Candidate Genes ◽  
Molecular Basis ◽  
Potential Candidate ◽  
New Findings

scholarly journals Truncation of the cytoplasmic tail of equine infectious anemia virus increases virion production by improving Env cleavage and plasma membrane localization

2021 ◽  
Author(s):  
Xue-Feng Wang ◽  
Yu-Hong Wang ◽  
Bowen Bai ◽  
Mengmeng Zhang ◽  
Jie Chen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Equine Infectious Anemia Virus ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Functional Domain ◽  
Live Attenuated Vaccine ◽  
Virion Production ◽  
Equine Infectious Anemia ◽  
Anemia Virus

Envelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence or absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C-terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT-truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication. Importance The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

scholarly journals The Potential of Candida Biofilm Protein as Bioreceptor for Candidiasis Immunoassay

2018 ◽  
Vol 14 (1) ◽  
pp. 47 ◽  
Author(s):  
Masfufatun Masfufatun ◽  
Loo Haryanto ◽  
Harsono Harsono
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Polyclonal Antibody ◽  
Control Group ◽  
Potential Candidate ◽  
Dot Blot ◽  
Protein Extract ◽  
Treatment Groups ◽  
Sds Page ◽  
Mechanical Methods

Abstract: Candidiasis or infection that is caused by Candida has become a new list of the therapeutical problems recently. The difficulties in diagnosing are the main cause of the unsatisfactory results from common therapies and diagnosis methods. This has urged researchers to find alternative ways in candidiasis diagnosis such as serology-based detection using antigen or antibody development. The aim of this study was to evaluate the potential of protein derived from Candida albicans biofilm as bioreceptor on candidiasis immunoassay through Dot Blot method. The research method used descriptive method with the following stages: (1) preparation of Candida albicans biofilm (2) extraction of Candida albicans protein through enzymatic and mechanical methods, (3) determination of protein molecular weight with SDS-PAGE (4) production of polyclonal anti- candida and (5) analysis of protein extract as bioreeceptor on dot blot. Profile of biofilm proteins on SDS-PAGE analysis were shown on molecular weight 27,42; 29,89; 38,10; 44,90; 48,75; 52,92; 55,14; 59,86; 70,56; 87,36; 102,54;115,05; 130,14;143,14;181,53 kD. There were differences in the intensity of dots in the control group (44070) and treatment groups (63170.5). It is noticeable that biofilm protein extract of C. albicans can be used for induction of anti-Candida polyclonal antibody production as the potential candidate of bioreceptor in candidiasis immunoassay. Keywords: SDS-PAGE, polyclonal antibody, immunoassay, dot blot, biofilm

scholarly journals The mechanism regulating the dissociation of the centrosomal protein C-Nap1 from mitotic spindle poles

2002 ◽  
Vol 115 (16) ◽  
pp. 3275-3284 ◽  
Author(s):  
Thibault Mayor ◽  
Ulrike Hacker ◽  
York-Dieter Stierhof ◽  
Erich A. Nigg
Keyword(s):  
Cell Cycle ◽  
Protein C ◽  
Proteasome Inhibitors ◽  
Full Length ◽  
Centrosomal Protein ◽  
Large Structures ◽  
Spindle Poles ◽  
M Phase ◽  
Nucleation Activity ◽  
Cycle Regulation

The centrosomal protein C-Nap1 is thought to play an important role in centrosome cohesion during interphase of the cell cycle. At the onset of mitosis, when centrosomes separate for bipolar spindle formation, C-Nap1 dissociates from centrosomes. Here we report the results of experiments aimed at determining whether the dissociation of C-Nap1 from mitotic centrosomes is triggered by proteolysis or phosphorylation. Specifically, we analyzed both the cell cycle regulation of endogenous C-Nap1 and the fate of exogenously expressed full-length C-Nap1. Western blot analyses suggested a reduction in the endogenous C-Nap1 level during M phase, but studies using proteasome inhibitors and destruction assays performed in Xenopus extracts argue against ubiquitin-dependent degradation of C-Nap1. Instead, our data indicate that the mitotic C-Nap1 signal is reduced as a consequence of M-phase-specific phosphorylation. Overexpression of full-length C-Nap1 in human U2OS cells caused the formation of large structures that embedded the centrosome and impaired its microtubule nucleation activity. Remarkably, however, these centrosome-associated structures did not interfere with cell division. Instead, centrosomes were found to separate from these structures at the onset of mitosis, indicating that a localized and cell-cycle-regulated activity can dissociate C-Nap1 from centrosomes. A prime candidate for this activity is the centrosomal protein kinase Nek2, as the formation of large C-Nap1 structures was substantially reduced upon co-expression of active Nek2. We conclude that the dissociation of C-Nap1 from mitotic centrosomes is regulated by localized phosphorylation rather than generalized proteolysis.

Comparative anlysis of the differential expressed proteins in serumbetween healthy and Brucellaovis infected sheep

2016 ◽  
Author(s):  
Guoping Wang ◽  
Run Wu
Keyword(s):  
Gel Electrophoresis ◽  
High Performance ◽  
Ion Trap ◽  
Serum Proteins ◽  
Differentially Expressed ◽  
High Abundance ◽  
Potential Candidate ◽  
Material Transport ◽  
Two Dimensional Gel Electrophoresis ◽  
Amyloid A

Disease-related serum proteins in sheep infected with Brucellaovis were assessed by conventional two-dimensional gel electrophoresis (2-DE). High-abundance proteins in serum were depleted by Albumin & IgG Depletion SpintrapTM, with the remaining proteins retrieved using the 2-D clean-up kit. Differentially expressed proteins were detected with the PDQUest8.0 software and identified by a high performance liquid chromatography system equipped with an ion trap mass spectrometer. Both SDS-PAGE and 2-DE gel electrophoresis confirmed that high-abundance protein components were removed thoroughly by the 2-D clean-up kit and Albumin & IgG Depletion SpintrapTM. A total of 9 differentially expressed protein spots were successfully identified as haptoglobin, serum amyloid A, Ig heavy chain, Igl-1 chain, and apolipoprotein C-III, known to be involved in stress response, energy metabolism, and material transport. These findings provide a valuable clue for investigating host immune response to pathogens at the protein level; further understanding of these novel proteins identified would be helpful for finding out potential candidate targets for diagnosis and developing new vaccines for brucellosis.

scholarly journals Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

2003 ◽  
Vol 77 (6) ◽  
pp. 3569-3577 ◽  
Author(s):  
Pamela J. Glass ◽  
Carl Q. Zeng ◽  
Mary K. Estes
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Capsid Protein ◽  
Full Length ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Orf3 Protein ◽  
Orf2 Protein

ABSTRACT Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF31-179, ORF31-152, and ORF31-107) no longer produced the 35K form. An N-terminal truncation mutant (ORF351-212) and a site-directed mutant (ORF3T201V) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3FL), ORF31-179, ORF31-152, and ORF351-212 interacted with the ORF2 protein, while an ORF31-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

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