The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone.

Ataxia telangiectasia (AT) is a rare genetic neurodegenerative disease. To date, there is no available cure for the illness, but the use of glucocorticoids has been shown to alleviate the neurological symptoms associated with AT. While studying the effects of dexamethasone (dex) in AT fibroblasts, by chance we observed that the nucleoplasmic Lamin A/C was affected by the drug. In addition to the structural roles of A-type lamins, Lamin A/C has been shown to play a role in the regulation of gene expression and cell cycle progression, and alterations in the LMNA gene is cause of human diseases called laminopathies. Dex was found to improve the nucleoplasmic accumulation of soluble Lamin A/C and was capable of managing the large chromatin Lamin A/C scaffolds contained complex, thus regulating epigenetics in treated cells. In addition, dex modified the interactions of Lamin A/C with its direct partners lamin associated polypeptide (LAP) 2a, Retinoblastoma 1 (pRB) and E2F Transcription Factor 1 (E2F1), regulating local gene expression dependent on E2F1. These effects were differentially observed in both AT and wild type (WT) cells. To our knowledge, this is the first reported evidence of the role of dex in Lamin A/C dynamics in AT cells, and may represent a new area of research regarding the effects of glucocorticoids on AT. Moreover, future investigations could also be extended to healthy subjects or to other pathologies such as laminopathies since glucocorticoids may have other important effects in these contexts as well.

NFE2L3 Inhibition Induces Cell Cycle Arrest at the G0/G1 Phase in Colorectal Cancer Cells through Downregulating CCND1 and pRb1-ser807/811.

The molecular mechanism for colorectal cancer to develop remains unelucidated. To find biomarkers related to colorectal cancer development, we analyzed the gene expression profile of 380 colorectal cancer patients and 51 healthy controls by R software. Finally, 1579 upregulated differential expression genes (DEGs) and 3218 downregulated DEGs were identified. Then, the top 20 upregulated DEGs were compared with 181 upregulated DEGs that we reported previously, and 11 overlapped DEGs were found. NFE2L3 (nuclear factor, erythroid 2-like 3) was among those overlapped DEGs and was rarely reported in colorectal cancer. Real-time polymerase chain reaction (PCR) results showed that higher NFE2L3 expression levels were identified in paired tumor samples than in paratumor samples (48 paired samples). Flow cytometry analysis revealed that the cell cycle was arrested at the G0/G1 phase after inhibition of NFE2L3 in both HCT116 and SW480 cell lines. Western blot detection showed that CCND1 and phosphorylated Rb transcriptional corepressor 1 at ser-807/811 (pRb1-ser807/811) expression levels were downregulated when NFE2L3 was inhibited in those two cell lines. A significant positive correlation was observed between NFE2L3 and CCND1 expression levels in colorectal tissue samples. These evidences indicate that downregulation of NFE2L3 induces cell cycle arrest at the G0/G1 phase through downregulation of CCND1 and pRb1-ser807/811.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

EZH2 is upregulated in the proliferation centers of CLL/SLL lymph nodes.

Lymph node involvement of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) is characterised by the diffuse infiltration of small neoplastic lymphocytes, which is accompanied by the presence of proliferation centres (PCs) comprising prolymphocytes and paraimmunoblasts. There is increasing evidence of accumulation of various molecular alterations in the tumour cells of PCs, which may explain why extended PCs are related to a less favourable prognosis. To further characterize PCs, we compared the expression level of EZH2 protein, the overexpression of which has recently been recognized as poor prognostic factor in CLL/SLL, in the PCs and the intervening small cell areas in lymph nodes of 15 patients with CLL/SLL. We also investigated the mutational profile of EZH2 and the expression of its upstream regulators c-Myc, E2F1, pRB and miR-26a. Our results showed a significantly increased expression of EZH2 in the PCs. No EZH2 mutations were detected, however, overexpression of c-Myc, E2F1 and pRb proteins as well as reduced expression of the tumor suppressor miR-26a were demonstrated in the PCs. In summary our findings indicate that EZH2 pathway is significantly upregulated in the PCs of CLL/SLL lymph nodes, providing further evidence for the distinguished biological features of the PCs.

The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

OBJECTIVES: The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. DESIGN: To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. RESULTS: The peptide sequences (I37PPPYSCTPNMNNCSR52, C53HHHHKRHHYPCNYCFCYPK72, R59HHYPCNYCFCYPK72 and H60HYPCNYCFCYPK72) present in the variant protein of P-B were identified. The peptide sequence (G6PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR36) in P-B (or the variant) and sequence (I37PPPPPAPYGPGIFPPPPPQP57) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. CONCLUSIONS: The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity.

Genetic- and Lifestyle-dependent Dental Caries Defined by the Acidic Proline-rich Protein Genes PRH1 and PRH2.

Dental caries is a chronic infectious disease that affects billions of people with large individual differences in activity. We investigated whether PRH1 and PRH2 polymorphisms in saliva acidic proline-rich protein (PRP) receptors for indigenous bacteria match and predict individual differences in the development of caries. PRH1 and PRH2 variation and adhesion of indigenous and cariogenic (Streptococcus mutans) model bacteria were measured in 452 12-year-old Swedish children along with traditional risk factors and related to caries at baseline and after 5-years. The children grouped into low-to-moderate and high susceptibility phenotypes for caries based on allelic PRH1, PRH2 variation. The low-to-moderate susceptibility children (P1 and P4a-) experienced caries from eating sugar or bad oral hygiene or infection by S. mutans. The high susceptibility P4a (Db, PIF, PRP12) children had more caries despite receiving extra prevention and irrespective of eating sugar or bad oral hygiene or S. mutans-infection. They instead developed 3.9-fold more caries than P1 children from plaque accumulation in general when treated with orthodontic multibrackets; and had basic PRP polymorphisms and low DMBT1-mediated S. mutans adhesion as additional susceptibility traits. The present findings thus suggest genetic autoimmune-like (P4a) and traditional life style (P1) caries, providing a rationale for individualized oral care.

Transcriptome analysis reveals molecular anthelmintic effects of procyanidins in C. elegans.

Worldwide, more than 1 billion people are affected by infestations with soil-transmitted helminths and also in veterinary medicine helminthiases are a severe threat to livestock due to emerging resistances against the common anthelmintics. Proanthocyanidins have been increasingly investigated for their anthelmintic properties, however, except for an interaction with certain proteins of the nematodes, not much is known about their mode of action. To investigate the anthelmintic activity on a molecular level, a transcriptome analysis was performed in Caenorhabditis elegans after treatment with purified and fully characterized oligomeric procyanidins (OPC). The OPCs had previously been obtained from a hydro-ethanolic (1:1) extract from the leaves of Combretum mucronatum, a plant which is traditionally used in West Africa for the treatment of helminthiasis, therefore, also the crude extract was included in the study. Significant changes in differential gene expression were observed mainly for proteins related to the intestine, many of which were located extracellularly or within cellular membranes. Among the up-regulated genes, several hitherto undescribed orthologues of structural proteins in humans were identified, but also genes that are potentially involved in the worms' defense against tannins. For example, T22D1.2, an orthologue of human basic salivary proline-rich protein (PRB) 2, and numr-1 (nuclear localized metal responsive) were found to be strongly up-regulated. Down-regulated genes were mainly associated with lysosomal activity, glycoside hydrolysis or the worms' innate immune response. No major differences were found between the groups treated with purified OPCs versus the crude extract. Investigations using GFP reporter gene constructs of T22D1.2 and numr-1 corroborated the intestine as the predominant site of the anthelmintic activity. The current findings support previous hypotheses of OPCs interacting with intestinal surface proteins and provide the first insights into the nematode's response to OPCs on a molecular level as a base for the identification of future drug targets.

In vitro cytotoxic potential of friedelin in human MCF-7 breast cancer cell: Regulate early expression of Cdkn2a and pRb1, neutralize mdm2-p53 amalgamation and functional stabilization of p53.

We aimed to explore the cytotoxic and apoptotic effect of friedelin on breast cancer MCF-7 cells. Cytotoxic effect of friedelin on MCF-7 cells was analyzed using MTT, cell and nuclear morphology. The apoptosis mechanism of friedelin on MCF-7 cells was analyzed using real-time PCR. Friedelin potentially inhibit 78% of MCF-7 cell's growth, the IC50 value was 1.8µM in 24h and 1.2µM in 48h. Friedelin increased ROS significantly and DNA damage was confirmed by tunel assay. We found characteristically 52% apoptotic cells and 6% necrotic cells in PI, AO/ErBr staining after 48h treatment with 1.2µM of friedelin. Apoptosis was confirmed by significantly (p≤0.001) increased tumor suppressor gene Cdkn1a, pRb2, p53, Nrf2, caspase-3 and decreased Bcl-2, mdm2 & PCNA expression after 48h. In conclusion, friedelin effectively inhibit breast cancer MCF-7 cell growth, it was associated with early expression of Cdkn1a, pRb2 and activation of p53 and caspases.

Zinc enhances CDKN2A, pRb1 expression and regulates functional apoptosis via upregulation of p53 and p21 expression in human breast cancer MCF-7 cell.

Zinc (Zn) is an essential trace elements, its deficiency is associated with increased incidence of human breast cancer. We aimed to study the effect of Zn on human breast cancer MCF-7 cells cultured in Zn depleted and Zn adequate medium. We found increased cancer cell growth in zinc depleted condition, further Zn supplementation inhibits the viability of breast cancer MCF-7 cell cultured in Zn deficient condition and the IC25, IC50 value for Zn is 6.2µM, 15µM, respectively after 48h. Zn markedly induced apoptosis through the characteristic apoptotic morphological changes and DNA fragmentation after 48h. In addition, Zn deficient cells significantly triggered intracellular ROS level and develop oxidative stress induced DNA damage; it was confirmed by elevated expression of CYP1A, GPX, GSK3ß and TNF-α gene. Zinc depleted MCF-7 cells expressed significantly (p≤0.001) decreased levels of CDKN2A, pRb1, p53 and increased the level of mdm2 expression. Zn supplementation (IC50=15µM), increased significantly CDKN2A, pRB1 & p53 and markedly reduced mdm2 expression; also protein expression levels of CDKN2A and pRb1 was significantly increased. In addition, intrinsic apoptotic pathway related genes such as Bax, caspase-3, 8, 9 & p21 expression was enhanced and finally induced cell apoptosis. In conclusion, physiological level of zinc is important to prevent DNA damage and MCF-7 cell proliferation via regulation of tumor suppressor gene.

Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.

Influence of clone and deletion size on outcome in chronic lymphocytic leukemia patients with an isolated deletion 13q in a population-based analysis in British Columbia, Canada.

Deletion of the long arm of chromosome 13 (del(13q)) as the sole abnormality in chronic lymphocytic leukemia (CLL) portends a good prognosis; however, there is great outcome heterogeneity within this subgroup. The percentage of cells with a del(13q) (clone size) and the extent of the deletion are two factors that may affect outcome in CLL patients with isolated del(13q). We analyzed 248 CLL patients from the BC Provincial CLL database identified as having isolated del(13q) detected pretreatment by interphase fluorescence in situ hybridization to determine what impact clone and deletion size had on overall survival (OS) and treatment free survival (TFS). Patients with 60% or more of nuclei with a del(13q) had shorter TFS and shorter OS. A large deletion, encompassing the RB1 gene locus, was detected in half of the 90 cases with available specimens for testing, and there was no significant difference in OS and TFS between RB1-deleted and RB1-not-deleted cases. Further study in a larger sample size is required to determine the clinical interest of RB1 locus testing; however, clone size of del(13q) does predict TFS and OS and may better refine prognosis in this clinically heterogeneous population.

The intriguing heterogeneity of human salivary proline-rich proteins: Short title: Salivary proline-rich protein species.

UNLABELLED: The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood. SIGNIFICANCE: The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals.

MLL fusion-driven activation of CDK6 potentiates proliferation in MLL-rearranged infant ALL.

Acute lymphoblastic leukemia in infants (< 1 year-of-age) is characterized by a high incidence of MLL rearrangements. Recently, direct targets of the MLL fusion protein have been identified. However, functional validation of the identified targets remained unacknowledged. In this study, we identify CDK6 as a direct target of the MLL fusion protein and an important player in the proliferation advantage of MLL-rearranged leukemia. CDK6 mRNA was significantly higher expressed in MLL-rearranged infant ALL patients compared with MLL wild-type ALL patients (P < 0.001). Decrease of MLL-AF4 and MLL-ENL fusion mRNA expression by siRNAs resulted in downregulation of CDK6, affirming a direct relationship between the presence of the MLL fusion and CDK6 expression. Knockdown of CDK6 itself significantly inhibited proliferation in the MLL-AF4-positive cell line SEM, whereas knockdown of the highly homologous gene CDK4 had virtually no effect on the cell cycle. Furthermore, we show in vitro sensitivity of MLL-rearranged leukemia cell lines to the CDK4/6-inhibitor PD0332991, inducing a remarkable G 1 arrest, and downregulation of its downstream targets pRB1 and EZH2. We therefore conclude that CDK6 is indeed a direct target of MLL fusion proteins, playing an important role in the proliferation advantage of MLL-rearranged ALL cells.

High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22 (Phos) → Phe variant.

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22 (phos)→Phe.

Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

Prolactinomas, or prolactin-secreting adenomas, constitute the most common type of hyperfunctioning pituitary adenoma. Dopamine agonists are used as first-line medication for prolactinomas, but the tumors are resistant to the therapy in 5-18 % of patients. To explore potential mechanisms of resistance to bromocriptine (a dopamine agonist), we analyzed six responsive prolactinomas and six resistant prolactinomas by whole-exome sequencing. We identified ten genes with sequence variants that were differentially found in the two groups of tumors. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in the 12 prolactinomas and in six normal pituitary glands. The mRNA levels of one of the genes, PRB3, were about fourfold lower in resistant prolactinomas than in the responsive tumors (p = 0.02). Furthermore, low PRB3 expression was also associated with tumor recurrence. Our results suggest that low levels of PRB3 mRNA may have a role in dopamine-agonist resistance and tumor recurrence of prolactinomas.

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes.

BACKGROUND: The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. RESULTS: We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. CONCLUSIONS: Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

An evolutionary perspective of mammal salivary peptide families: cystatins, histatins, statherin and PRPs.

Saliva's role in the oral cavity, such as lubrication, protection of tissues and antimicrobial action is a reflex of its composition, among which are several peptide families like statherin, histatins, proline rich proteins (PRPs) and some members of the cystatin family. These peptides present different evolutionary pathways being in the case of histatin, statherin and PRP families restricted to few millions and comprising few species when compared with cystatins, where duplication occurred at more than 650 mya. Though the recognized relevance of phylogenetic approaches to disclose relationships among different species, information on the salivary proteins that allow the association between peptide families-related structure and function in the oral cavity is scarce. In the present study, the four major salivary peptides classes are reviewed considering the few known phylogenetic studies focusing on their evolution among mammals. New perspectives and challenges for future and multidisciplinary experimental works are drawn.

MYCN-mediated overexpression of mitotic spindle regulatory genes and loss of p53-p21 function jointly support the survival of tetraploid neuroblastoma cells.

High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells.

Expression of PRB, FKBP52 and HB-EGF relating with ultrasonic evaluation of endometrial receptivity.

BACKGROUND: To explore the molecular basis of the different ultrasonic patterns of the human endometrium, and the molecular marker basis of local injury. METHODOLOGY/PRINCIPAL FINDINGS: The mRNA and protein expression of FKBP52, progesterone receptor A (PRA), progesterone receptor B (PRB), and HB-EGF were detected in different patterns of the endometrium by real-time RTPCR and immunohistochemistry. There were differences in the mRNA and protein expression of FKBP52, PRB, and HB-EGF in the triple line (Pattern A) and homogeneous (Pattern C) endometrium in the window of implantation. No difference was detected in PRA expression. After local injury, the mRNA expression of HB-EGF significantly increased. In contrast, there was no difference in the mRNA expression of FKBP52, PRB, or PRA. The protein expression of FKBP52, PRB, and HB-EGF increased after local injury. There was no difference in the PRA expression after local injury. CONCLUSIONS: PRB, FKBP52, and HB-EGF may be the molecular basis for the classification of the ultrasonic patterns. HB-EGF may be the molecular basis of local injury. Ultrasonic evaluation on the day of ovulation can be effective in predicting the outcome of implantation.

Finding new posttranslational modifications in salivary proline-rich proteins.

Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC-MALDI-TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N-glycosylation sites: two in basic proline-rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O-glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N-terminal pyro-Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP-modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro-Glu. Moreover, comparing all subject groups, it was noticed a predominance of N-acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients.