Tris-Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass.

Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

The Treatment Landscape and New Opportunities of Molecular Targeted Therapies in Gastroenteropancreatic Neuroendocrine Tumors.

Neuroendocrine neoplasms (NENs) are a heterogeneous group of neoplasms that originate from neuroendocrine stem cells and express both neural and endocrine markers. They are found in almost every organ, and while NENs are mostly associated with slow growth, complications due to the uncontrolled secretion of active peptides, and metastatic disease, may significantly impair the quality of life and can ultimately lead to the death of affected individuals. Expanding knowledge of the genetic, epigenetic, and proteomic landscapes of NENs has led to a better understanding of their molecular pathology and consequently increased treatment options for patients. Here, we review the principal breakthroughs in NEN treatment management, owing largely to omics technologies over the last few years, current recommendations of systemic treatment, and ongoing research into the identification of predictive and response biomarkers based on molecular targeted therapies.

The E3 ubiquitin protein ligase HERC2 modulates the activity of tumor protein p53 by regulating its oligomerization.

The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292-2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.

Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass.

Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and that it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-acetate polyacrylamide gels.

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3-15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris-acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris-Tricine-SDS-sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS-PAGE such as CBB staining and immunoblot. Thus, by using Tris-acetate 3-15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10-500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), beta-actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 microg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G<-->A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.