Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

Background

Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.

Results

The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.

Conclusions

In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.     

Cloning and Characterization of a Novel, Human Cellular Retinaldehyde-binding Protein CRALBP-like (CRALBPL) Gene

Cellular retinaldehyde-binding protein (CRALBP) plays a role in the vertebrate visual process as a substrate-routing protein. It belongs to a widespread lipid-binding SEC14-like protein family. All the members of the family have the lipid-binding domain called CRAL-TRIO. Here we have isolated a new human CRAL-TRIO domain containing a CRALBP-like (CRALBPL) gene from the cDNA library of human adult brain. The CRALBPL gene consisted of 1,694 bp and had an ORF encoding putatively 354 amino acids with a CRAL-TRIO domain from 118 to 279 aa. The expression pattern in 18 human tissues indicated that CRALBPL gene was mainly expressed in brain. The alignment of CRAL-TRIO domain showed that CRALBPL had 45% identity with human CRALBP. Subcellular location revealed that CRALBPL protein was located in the cytoplasm of HeLa cells. Western blotting indicated that the CRALBPL had a molecular weight of about 40 kDa.     

PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis

Paraneoplastic Ma Family (PNMA) comprises a growing number of family members which share relatively conserved protein sequences encoded by the human genome and is localized to several human chromosomes, including the X-chromosome. Based on sequence analysis, PNMA family members share sequence homology to the Gag protein of LTR retrotransposon, and several family members with aberrant protein expressions have been reported to be closely associated with the human Paraneoplastic Disorder (PND). In addition, gene mutations of specific members of PNMA family are known to be associated with human mental retardation or 3-M syndrome consisting of restrictive post-natal growth or dwarfism, and development of skeletal abnormalities. Other than sequence homology, the physiological function of many members in this family remains unclear. However, several members of this family have been characterized, including cell signalling events mediated by these proteins that are associated with apoptosis, and cancer in different cell types. Furthermore, while certain PNMA family members show restricted gene expression in the human brain and testis, other PNMA family members exhibit broader gene expression or preferential and selective protein interaction profiles, suggesting functional divergence within the family. Functional analysis of some members of this family have identified protein domains that are required for subcellular localization, protein-protein interactions, and cell signalling events which are the focus of this review paper.     

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.     

Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family

S100 proteins are low-molecular-weight calcium-binding proteins of the EF-hand superfamily and appear to be involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. More than 10 members of the S100 protein family have been described from human sources so far. We have now isolated a YAC clone from human chromosome 1q21, on which 9 different genes coding for S100 calcium-binding proteins could be localized. Moreover, we have mapped the gene coding for S100P to human chromosome 4p16 and thereby completed the chromosomal assignments of all known human S100 genes. The clustered organization of S100 genes in the 1q21 region allows us to introduce a new logical nomenclature for these genes, which is based on the physical arrangement on the chromosome. The new nomenclature should facilitate and further the understanding of this protein family and be easily expandable to other species.     

A proteomic approach to study parathyroid glands

Parathyroid tumours are heterogeneous and in some cases the diagnosis may be difficult using histological features. In this study we used a two-dimensional electrophoresis (2D)/mass spectrometry (MS)-based approach to examine the global changes of parathyroid adenoma tissues protein profile compared to the parathyroid normal tissues. Validation of protein expression was performed by immunoblotting using specific antibodies. Ingenuity software was used to identify the biological processes to which these proteins belong and to construct a potential network. A total of 30 proteins were found to be differentially expressed, of which 22 resulted in being over-expressed. Proteins identified by 2D/MS/MS proteomics were classified into functional categories and a major change (≥ 2-fold) in terms of expression was found in proteins involved in response to biotic stimuli, cell organization and signal transduction. After Ingenuity analysis, 14-3-3 ζ/δ appears to be a key protein in the network of parathyroid adenoma, where it is linked to other proteins such as annexin A2, B box and SPRY domain-containing protein (BSPRY), p53 and epidermal growth factor receptor (EGFR). Our results suggest that the proteomic approach was able to differentiate the protein profiles of normal parathyroid and parathyroid adenoma and identify a panel of proteins which are differentially expressed. The functional role of these proteins in the network of intracellular pathways is discussed.     

A hitchhiker's guide to the human Hsp70 family

The human Hsp70 family encompasses at least 11 genes which encode a group of highly related proteins. These proteins include both cognate and highly inducible members, at least some of which act as molecular chaperones. The location of cognate Hsp70s within all the major subcellular compartments is an indication of the importance of these proteins. The expression of several inducible Hsp70 genes is also an indication of the importance of these proteins in the stres response. The existence of multiple genes and protein isoforms has created confusion in the identification and naming of particular family members. We have compiled, from the literature, a list of genes and genetic loci and produced a two-dimensional protein map of the known human Hsp70 family members. This will enable researchers in the field to quickly and reliably identify human Hsp70s. We have also devised a more rational nomenclature for these genes and gene products which, subject to general acceptance, could be extended to Hsp70 families from other species.     

The cloning and nucleotide sequence of human ST2L cDNA

The ST2 gene is a member of the IL-1 receptor family and is hypothesized to be involved in helper T cell function, but its functional ligand and physiological role remain unknown. We have cloned the human ST2L cDNA that encodes a distinct type of membrane-bound ST2 protein. The predicted 556-amino-acid sequence showed 67% identity to the mouse ST2L protein. The human ST2 gene (IL1RL1) contains 13 exons and spans 40 kb in length. Its exon-intron organization was elucidated from a registered human genomic sequence derived from chromosome 2q, which contains three other genes belonging to the IL-1 receptor family in an approximately 202-kb genomic region. The tissue distribution of ST2 expression was examined by RT-PCR, and the soluble form (ST2, IL1RL1-a) and ST2L (IL1RL1-b) appear to be expressed differentially. We also established stable transfectants of a human glioblastoma cell line, T98G, that express human ST2L constitutively, and we confirmed cell-surface expression of human ST2L protein on the transfectants.     

Molecular cloning and expression of a human chondroitin synthase

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.     

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.     

Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18)

The ubiquitin-specific proteases (UBP) are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates. We have recently cloned UBP43, a novel member of this family from AML1-ETO knock-in mice. To analyze the role of UBP43 in hematopoiesis and leukemogenesis, we have cloned a full-length human UBP43 cDNA by screening a human monocytic cDNA library as well as by 5'- and 3'-rapid amplification of cDNA ends analyses. This cDNA encodes a polypeptide of 372 amino acids with all of the structural motifs of a deubiquitinating enzyme. The human UBP43 mRNA is strongly expressed in human liver and thymus. Transfection analysis has demonstrated that UBP43 is a nuclear protein. Interestingly, the gene encoding human UBP43 maps to chromosome 22q11.2. This region, known as DiGeorge syndrome critical region, contains a minimal area of 2 Mb and is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by thymic aplasia or hypoplasia, parathyroid hypoplasia, or congenital cardiac abnormalities. Taken together, our results broaden the understanding of a new human ubiquitin-specific protease, UBP43, and suggest that this gene may also be related to DiGeorge syndrome.     

Bone morphogenetic protein 4 (BMP-4), a member of the TGF-beta family, in early embryos of Xenopus laevis: analysis of mesoderm inducing activity

We have screened a Xenopus ovary cDNA library using a synthetic oligonucleotide derived from that part of the inhibin beta A sequence, which is highly conserved within the TGF-beta family. Out of several clones yielding autoradiographic signals four turned out to represent Xenopus counterparts to the human bone morphogenetic protein 4 (BMP-4). Each two of the four sequences are nearly identical and probably account for different alleles whereas the two pairs showing 5% divergence may have arisen by genome duplication in this tetraploid species. The amino acid sequence of the Xenopus protein is 80% homologous to the human sequence showing no single exchange within the last 100 amino acids at the C-terminus. This region, which constitutes the main part of the mature, biologically active protein, also exhibits substantial homologies to other representatives of the TGF-beta family, especially to the Drosophila DPPC protein. Transfection of COS-1 cells with the Xenopus BMP-4 sequence under control of the CMV-promoter leads to the secretion of a protein which exhibits mesoderm inducing activity when tested with animal cap explants from Xenopus blastula stage embryos.     

Expression of a novel member of sorting nexin gene family,SNX-L,in human liver development

The sorting nexin (SNX) protein family is implicated in the regulation of receptor degradation and membrane traffic in the cell. With the aim of identifying novel genes involved in receptor degradation and recycling, we have cloned a new member of the sorting nexin gene family, human sorting nexin L, SNX-L (or SNX21). This gene includes 4 exons and 3 introns, and is located on chromosome 20q12-13.1 region, encompassing 8 kb. The full-length cDNA of SNX-L is 1,811 bp, with an open reading frame of 1,092 bp. The protein consists of 364 amino acids and encodes a 40 kDa protein. The SNX-L protein has a common PX domain shared by all SNX family members. The similarity of SNX-L PX domain to the PX consensus sequence is over 40%. PX domains have been shown to associate with specific phospholipids and membrane compartments. Expression analysis of SNX-L mRNA indicates that SNX-L is distinctly and highly expressed in fetus liver, but only weakly expressed in brain, muscle (skeleton muscle, smooth muscle, and cardiac muscle), kidney, and adrenal gland. Strong liver expression of SNX-L is maintained from 12 to 25 weeks during human fetus development, suggesting that SNX-L may be a regulatory gene involved in receptor protein degradation during embryonic liver development.     

Structure of the human retinoblastoma-related p107 gene and its intragenic deletion in a B-cell lymphoma cell line

The human p107 protein shares many structural and functional features with the retinoblastoma gene product and retinoblastoma-related p130 protein. In this study, we have cloned and elucidated the complete intron-exon organization of the gene encoding the p107 protein. The gene contains 22 exons spanning over 100kilobase pairs of genomic DNA. The length of individual exons ranges from 50 to 840base pairs. The arrays of exons in the p107 gene are rather similar among members of the gene family, especially to those of the p130 gene, while the length of introns is extensively diverse. This study will provide a molecular basis for implementing comprehensive screening for p107 mutations using genomic DNAs from human malignancies.We also show a detailed structure of an intragenic deletion of the p107 gene found in a human B-cell lymphoma cell line, KAL-1, which was shown to occur by homologous recombination between the two directly repeated Alu family sequences.     

Substrate recognition by the protein disulfide isomerases

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate-limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol-disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate-binding sites and on determining their natural substrates in vivo.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

Isolation,characterization and molecular 3D model of human GDE4, a novel membrane protein containing glycerophosphodiester phosphodiesterase domain

As a transmembrane protein family, glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. To date, seven mammalian GDEs have been virtually cloned or predicted by bioinformatics analysis, however, GDE4 has not been molecular isolated and characterized in mammal. Here we report molecular cloning of human GDE4 encoding cDNA sequence, which is 945 base pairs long encoding a 314-amino acid protein with 2 transmembrane regions and a GDE motif. The human GDE1 gene is located on chromosome 19q22 and contains ten exons and nine introns. A molecular 3-D model provides the first structural information of human GDE4 and suggests a triose-phosphate-isomerase barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic residues highlights that the individual core residues Glu72, Asp74, and His87 are crucial to maintaining GDE4 catalytic activity. Western blotting shows that human GDE4 is a 36 kDa protein. Subcellular localization of GDE4 tagged with enhanced green fluorescence protein is in the cytoplasm, especially accumulated in the perinuclear region and the cell periphery. Moreover, over-expression of GDE4 did not induce neurite formation or change cell morphology. These results indicate GDE4 protein is a member of the GDE family and suggest it may play different roles from other members of GDE family.