Chaperone Therapy for GM2 Gangliosidosis: Effects of Pyrimethamine on β-Hexosaminidase Activity in Sandhoff Fibroblasts

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from β-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of β-hexosaminidase activity in cultured fibroblasts from Sandhoff patients.     

Aquaporins in cancer

Background

The aquaporins (AQPs) are a family of 13 small hydrophobic integral transmembrane water channel proteins involved in transcellular and transepithelial water movement, transport of fluid and cell migration.

Scope of the review

This review article summarizes our knowledge concerning the involvement of AQPs in tumor growth, angiogenesis and metastatic process.

Major conclusions

Tumor cells types express AQPs and a positive correlation exists between histological tumor grade and the AQP expression. Moreover, AQPs are involved also in tumor edema formation and angiogenesis in several solid and hematological tumors.

General significance

AQPs inhibition in endothelial and tumor cells might limit tumor growth and spread, suggesting a potential therapeutic use in the treatment of tumors. This article is part of a Special Issue entitled Aquaporins.     

Renal aquaporins and water balance disorders

Background

Aquaporins (AQPs) are a family of proteins that can act as water channels. Regulation of AQPs is critical to osmoregulation and the maintenance of body water homeostasis. Eight AQPs are expressed in the kidney of which five have been shown to play a role in body water balance; AQP1, AQP2, AQP3, AQP4 and AQP7. AQP2 in particular is regulated by vasopressin.

Scope of review

This review summarizes our current knowledge of the underlying mechanisms of various water balance disorders and their treatment strategies.

Major conclusions

Dysfunctions of AQPs are involved in disorders associated with disturbed water homeostasis. Hyponatremia with increased AQP levels can be caused by diseases with low effective circulating blood volume, such as congestive heart failure, or osmoregulation disorders such as the syndrome of inappropriate secretion of antidiuretic hormone. Treatment consists of fluid restriction, demeclocycline and vasopressin type-2 receptor antagonists. Decreased AQP levels can lead to diabetes insipidus (DI), characterized by polyuria and polydipsia. In central DI, vasopressin production is impaired, while in gestational DI, levels of the vasopressin-degrading enzyme vasopressinase are abnormally increased. Treatment consists of the vasopressin analogue dDAVP. Nephrogenic DI is caused by the inability of the kidney to respond to vasopressin and can be congenital, but is most commonly acquired, usually due to lithium therapy. Treatment consists of sufficient fluid supply, low-solute diet and diuretics.

General significance

In recent years, our understanding of the underlying mechanisms of water balance disorders has increased enormously, which has opened up several possible new treatment strategies. This article is part of a Special Issue entitled Aquaporins.     

Human aquaporins: Regulators of transcellular water flow

Background

Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistent with their expression in most tissues, AQPs are associated with diverse physiological and pathophysiological processes.

Scope of review

AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volume regulation (CVR) is particularly notable. This review examines the regulatory role of AQPs in transcellular water flow, especially in CVR. We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema.

Major conclusions

AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapid movement of water across diverse cell membranes and playing regulatory roles in CVR. Gating mechanisms have been proposed for human AQPs, but have only been reported for plant and microbial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow.

General significance

Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.     

The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach

Protein glycation is initiated by a nucleophilic addition reaction between the free amino group from a protein, lipid or nucleic acid and the carbonyl group of a reducing sugar. This reaction forms a reversible Schiff base, which rearranges over a period of days to produce ketoamine or Amadori products. The Amadori products undergo dehydration and rearrangements and develop a cross-link between adjacent proteins, giving rise to protein aggregation or advanced glycation end products (AGEs). A number of studies have shown that glycation induces the formation of the β-sheet structure in β-amyloid protein, α-synuclein, transthyretin (TTR), copper-zinc superoxide dismutase 1 (Cu, Zn-SOD-1), and prion protein. Aggregation of the β-sheet structure in each case creates fibrillar structures, respectively causing Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and prion disease. It has been suggested that oligomeric species of glycated α-synuclein and prion are more toxic than fibrils. This review focuses on the pathway of AGE formation, the synthesis of different types of AGE, and the molecular mechanisms by which glycation causes various types of neurodegenerative disease. It discusses several new therapeutic approaches that have been applied to treat these devastating disorders, including the use of various synthetic and naturally occurring inhibitors. Modulation of the AGE-RAGE axis is now considered promising in the prevention of neurodegenerative diseases. Additionally, the review covers several defense enzymes and proteins in the human body that are important anti-glycating systems acting to prevent the development of neurodegenerative diseases.     

Induction of Apoptosis in Human Hepatocarcinoma SMMC-7721 Cells In Vitro by Psoralen from Psoralea corylifolia

Psoralen is a major active component of Psoralea corylifolia. In the present study, we analyzed psoralen-induced changes in human hepatocarcinoma cell viability and apoptosis, and investigated the underlying mechanisms of the proapoptotic effect of the compound on SMMC-7721 cells. We measured human hepatocarcinoma cell viability by MTT assay and Annexin V-FITC/PI double staining, and evaluated the activity of caspase 3 and the expression of p53, Bax, and Bcl-2 proteins, involved in regulating cell apoptosis. Psoralen was able to inhibit the growth of SMMC-7721 cells in a dose- and time-dependent manner and had a strong proapoptotic effect on these cells. We show a dose-dependent increase in caspase-3 activity, and elevated levels of p53 and Bax proteins in psoralen-treated cells, that coincided with dose-dependent decrease in Bcl-2 expression. These results suggest that psoralen induces apoptosis in cancer cells via mechanisms that involve caspase-3, p53, Bax, and Bcl-2 pathway. Our results may provide a molecular basis for the further development of natural compounds as novel anticancer agents for human hepatomas.     

Aquaporin and brain diseases

Background

The presence of water channel proteins, aquaporins (AQPs), in the brain led to intense research in understanding the underlying roles of each of them under normal conditions and pathological conditions.

Scope of review

In this review, we summarize some of the recent knowledge on the 3 main AQPs (AQP1, AQP4 and AQP9), with a special focus on AQP4, the most abundant AQP in the central nervous system.

Major conclusions

AQP4 was most studied in several brain pathological conditions ranging from acute brain injuries (stroke, traumatic brain injury) to the chronic brain disease with autoimmune neurodegenerative diseases. To date, no specific therapeutic agents have been developed to either inhibit or enhance water flux through these channels. However, experimental results strongly underline the importance of this topic for future investigation. Early inhibition of water channels may have positive effects in prevention of edema formation in brain injuries but at later time points during the course of a disease, AQP is critical for clearance of water from the brain into blood vessels.

General significance

Thus, AQPs, and in particular AQP4, have important roles both in the formation and resolution of edema after brain injury. The dual, complex function of these water channel proteins makes them an excellent therapeutic target. This article is part of a Special Issue entitled Aquaporins.     

A Comparative Protein Profile of Mammalian Erythrocyte Membranes Identified by Mass Spectrometry

A comparative analysis of erythrocyte membrane proteins of economically important animals, goat (Capra aegagrus hircus), buffalo (Bubalus bubalis), pig (Sus scrofa), cow (Bos tauras), and human (Homo sapiens) was performed. Solubilized erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE), visualized by staining the gels with Commassie Brilliant Blue (CBB), and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Emerging results show that all major erythrocyte membrane proteins present in human are also seen in all the animals except for band 4.5 which could not be identified. Band 3 is seen as more intense and compact, band 4.1 appears as a doublet in all the animal erythrocyte membranes, band 4.2 exhibits a slightly higher molecular weight (Mr) in buffalo, and cow and band 4.9 has a higher Mr in all the animals relative to the human protein. In addition, there are two new bands in the goat membrane, band G1, identified as HSP 90α, and band G2 identified as HSP 70. A new band C2 identified as HSP 70 is also seen in cow membranes. Peroxiredoxin II is of lower intensity and/or higher Mr in the animals. The difference in size of the proteins possibly indicates the variations in the composition of the amino acids. The difference in intensity of the proteins among these mammalians highlights the presence of less or more number of copies of that protein per cell. This data complement the earlier observations of differences in the sialoglycoprotein profile and effect of proteases and neuraminidase on agglutination among the mammalian erythrocytes. This study provides a platform to understand the molecular architecture of the individual erythrocytes, and in turn the dependent disorders, their phylogenetic relationship and also generates a database of erythrocyte membrane proteins of mammals. The animals selected for this study are of economic importance as they provide milk for the dairy industry and raw material for leather industry and are routinely sacrificed to obtain non vegetarian food worldwide.     

Negative Purifying Selection Drives Prion and Doppel Protein Evolution

The prion protein (PrP) when misfolded into the pathogenic conformer PrP^Sc is the major causative agent of several lethal transmissible spongiform encephalopathies in mammals. Studies of evolutionary pressure on the corresponding gene using different datasets have yielded conflicting results. In addition, putative PrP or PrP interacting partners with strong similarity to PrP such as the doppel protein have not been examined to determine if the same evolutionary mechanisms apply to prion paralogs or if there are coselected sites that might indicate how and where the proteins interact. We examined several taxonomic groups that contain model organisms of prion diseases focusing on primates, bovids, and an expanded dataset of rodents for selection pressure on the prion gene (PRNP) and doppel gene (PRND) individually and for coevolving sites within. Overall, the results clearly indicate that both proteins are under strong selective constraints with relaxed selection on amino acid residues connecting α-helices 1 and 2.     

ATP significantly increases P2Y2-dependent RANTES secretion and overexpression in human airway epithelial cells

Uncontrolled inflammation is frequently observed in human respiratory diseases. Extracellular ATP can induce a number of physiological phenomena via binding to purinergic receptors. In spite of the fact that ATP has long been known as a proinflammatory mediator in the airway, the signaling pathway mechanism is still unclear. Here we show that ATP increases RANTES secretion and overexpression in a time-dependent manner and siRNA-P2Y₂ significantly decreases RANTES secretion and overexpression. These results suggest that ATP can induce secretion and overexpression of the RANTES chemokine via a P2Y₂ Gαq coupled receptor-dependent manner. In addition, pharmacological inhibition of ERK1/2 MAPK by U0126 suppressed ATP/P2Y₂-induced RANTES overexpression in the human airway epithelium. These results show that RANTES secretion and overexpression are regulated by a P2Y₂ receptor and the ATP/P2Y₂ signaling complex may be critical for airway inflammation in respiratory diseases. Taken together, our investigation provides novel insight into the physiological functions of the P2Y₂ receptor and enhances our understanding of the inflamed microenvironment in the airway.     

Feasibility study of gel filtration as a separation method for blood serum proteins in combination with PIXE analysis

The combination between a protein separation technique and the PIXE method has a great potential for large surveys, including thousands of samples, in which multielemental analysis is required. Gel filtration with a Sephadex G-200 gel and a TRIS-acetate buffer was used for separating proteins in human serum. The fractions were doped with an yttrium/vanadium standard and then concentrated and pipeted onto Kimfol^? backing foils. Using the PIXE technique, the distributions of Fe, Cu, Zn, and Se, with respect to molecular size, were found, indicating binding to specific proteins. Sulfur and phosphorus were found to correlate well with the protein content measured by UV-absorption at 280 nm. Further developments and tests on the protein separation technique is required, taking restrictions imposed by the PIXE method into consideration.     

Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the human cancer protein interaction network

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30 % sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein–protein interactions.     

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.     

The separation and comparison of albumin complexes of seed proteins in three cultivars ofPhaseolus vulgaris

Complexes of water soluble proteins (albumins) were investigated in three cultivarsof Phaseolus vulgaris, viz: Yeltruská Saxa, Vainica Saavegra B, and Krupnaya sakharnaya. The first two cultivars exhibit haemagglutinating activity against rabbit erythrocytes, but have different elution profiles on Sephadex G-100. Their individual peaks have a different subunit composition, as revealed by SDS gel electrophoresis, as well as a different immunoelectrophoretic pattern, although proteins I and II of the specificity Veltruská Saxa are present in both cultivars. The cultivar Krupnaya sakharnaya expressively differs from the preceding lectin cultivars; it has no erythroagglutinating activity, its albumin complex has a high-molecular component, absent in the preceding ones, and has no lectin peak in the region of molecular mass of 100 000 to 200 000. Immunoelectrophoresis gave no evidence of protein I and II of the specificity Veltruská Saxa.