Pawan Puri

Proteomic Analysis of Bovine Sperm YWHA Binding Partners Identify Proteins Involved in Signaling and Metabolism

Abstract Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.

Selective Ablation of Ppp1cc Gene in Testicular Germ Cells Causes Oligo-Teratozoospermia and Infertility in Mice

ABSTRACT The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (−/−) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

The Tyrosine Phosphatase SHP2 Regulates Sertoli Cell Junction Complexes

ABSTRACT The blood-testis barrier (BTB) is a large junctional complex composed of tight junctions, adherens junctions, and gap junctions between adjacent Sertoli cells in the seminiferous tubules of the testis. Maintenance of the BTB as well as the controlled disruption and reformation of the barrier is essential for spermatogenesis and male fertility. Tyrosine phosphorylation of BTB proteins is known to regulate the integrity of adherens and tight junctions found at the BTB. SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) and a key regulator of growth factor-mediated tyrosine kinase signaling pathways. We found that SHP2 is localized to Sertoli-Sertoli cell junctions in rat testis. The overexpression of a constitutive active SHP2 mutant, SHP2 Q79R, up-regulated the BTB disruptor ERK1/2 via Src kinase in primary rat Sertoli cells in culture. Furthermore, focal adhesion kinase (FAK), which also supports BTB integrity, was found to interact with SHP2 and constitutive activation of SHP2 decreased FAK tyrosine phosphorylation. Expression of the SHP2 Q79R mutant in primary cultured Sertoli cells also resulted in the loss of tight junction and adherens junction integrity that corresponded with the disruption of the actin cytoskeleton and mislocalization of adherens junction and tight junction proteins N-cadherin, β-catenin, and ZO-1 away from the plasma membrane. These results suggest that SHP2 is a key regulator of BTB integrity and Sertoli cell support of spermatogenesis and fertility.

The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase

SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit‐amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor‐mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis. Stem Cells 2014;32:741–753

Phosphorylation-Dependent Interaction of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein (YWHA) with PADI6 Following Oocyte Maturation in Mice

Abstract Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation.

Identification of testis 14–3-3 binding proteins by tandem affinity purification

The 14–3-3 family of proteins interacts with various cellular phosphoproteins and regulates multiple cell signaling cascades. Identification of 14–3-3 interactors is important to define 14–3-3 functions in various biological pathways. The binding partners of protein 14–3-3 in testis are not known. The main goal of this study was to identify the 14–3-3 interactome in testis to determine the 14–3-3 regulated cellular processes in testis. We used transgenic mice expressing tandem affinity tagged 14–3-3ζ (TAP-14–3-3ζ) driven by the ubiquitin promoter to isolate 14–3-3 binding proteins. The 14–3-3 complexes in testis were isolated using a two-step tandem affinity purification (TAP) followed by identification with liquid chromatography/tandem mass spectrometry (LC-MS/MS). A total of 135 proteins were found to be associated with 14–3-3 in vivo in testis. Comparison of the testis 14–3-3 proteome with known 14–3-3 binding proteins showed that 71 of the proteins identified in this study are novel 14–3-3 interactors. Eight of these novel 14–3-3 interacting proteins are predominantly expressed in testis. The 14–3-3 interactors predominant in testis are: protein phosphatase1γ2 (PP1γ2), spermatogenesis associated 18 (SPATA18), phosphoglycerate kinase-2 (PGK2), testis specific gene A-2 (TSGA-2), dead box polypeptide 4 (DDX4), piwi homolog 1, protein kinase NYD-SP25, and EAN57. The fact that some of these proteins are indispensable for spermatogenesis suggests that their binding to 14–3-3 may be important for their function in germ cell division and maturation. These findings are discussed in context of the putative functions of 14–3-3 in spermatogenesis.

The regulation of male fertility by the PTPN11 tyrosine phosphatase.

PTPN11 (also known as SHP2) is a ubiquitously expressed non-receptor tyrosine phosphatase that regulates cell survival, proliferation, differentiation, migration and adhesion. Naturally occurring mutations in the PTPN11 gene cause Noonan and LEOPARD syndromes, two genetic disorders that are characterized by a spectrum of defects including male infertility. This review summarizes four cellular and molecular mechanisms by which PTPN11 acts to support male fertility. First, PTPN11 is required for the proliferation and survival of spermatogonial stem cells (SSCs) that are essential to replenish the germ cells that will become sperm. Second, PTPN11 regulation of cellular adhesion functions in Sertoli cells is required to maintain the blood-testis barrier (BTB) that protects meiotic and post-meiotic germ cells. Third, expression of PTPN11 in Sertoli cells is essential to prevent premature differentiation and exhaustion of the SSC population and to maintain the SSC niche. Finally, in Leydig cells, PTPN11 supports mitochondrial fusion and the expression of acyl-CoA synthetase (ACSL4) needed for the production of steroids including testosterone, which is required for fertility.

Six2creFrs2α knockout mice are a novel model of renal cystogenesis

Six2cre-mediated deletion of Frs2α (Six2creFrs2αKO), a major fibroblast growth factor receptor (Fgfr) docking protein in mouse nephron progenitors results in perinatal renal hypoplasia; however, postnatal Six2creFrs2αKO kidneys develop cysts. We sought to determine the pathogenesis of Six2creFrs2αKO cyst formation. We performed histological assays, Western blots, and quantitative PCR (qPCR). While embryonic day (E) 18.5 Six2Frs2αKO kidneys were hypoplastic and not cystic, postnatal day (P) 7 mutants had proximal tubular-derived cysts that nearly replaced the renal parenchyma by P21. Mutants had high proximal tubular proliferation rates and interstitial fibrosis, similar to known polycystic kidney disease (PKD) models. Six2creFrs2αKO kidneys also had upregulation of Wnt/βcatenin signaling, macrophage infiltration and chemokine production (e.g. ectopic Ccl2 in non-dilated proximal tubules), and augmented hedgehog signaling, features also seen in other PKD models. We saw increased Gli1 (hedgehog readout) in postnatal Six2creFrs2αKO interstitium and ectopic sonic hedgehog (Shh) in subsets of non-dilated P7 mutant proximal tubules (likely driving the stromal Gli expression). As ectopic tubular Shh and Ccl2 expression is seen after acute kidney injury (AKI), we interrogated another bone fide AKI marker, Kim1 and noted ectopic expression in P7 non-dilated proximal tubules. These observations suggest that aberrantly activated “AKI” pathways may drive pathogenesis in PKD.

Renal Development and Molecular Pathogenesis of Renal Dysplasia

Abstract Renal dysplasia is a leading cause of chronic kidney disease and end stage kidney disease in children. Understanding the molecular pathogenesis of renal maldevelopment began in the 1950s with classical studies by Clifford Grobstein, who isolated and characterized primordial tissues that interact to form the metanephric (adult) kidney. Since then, studies have shown that the following four embryonic tissues develop and send reciprocal signaling to form the kidney: the nephrogenic mesenchyme, the ureteric bud, the stromal mesenchyme, and the renal vasculature. Molecular biology and gene targeting techniques have also identified many genes and signaling pathways required for proper kidney formation in animal models. Recently epigenetic mechanisms have also been described as necessary for renal development in animal models. Studies in patients with syndromic and nonsyndromic renal dysplasia and other congenital renal malformations have identified many causative genetic defects that were previously identified in animal models. This chapter first reviews the molecular control of the embryonic tissues that form the kidney and then discusses the genes known to contribute to renal dysplasia in patients.

Ectopic Phosphorylated Creb Marks Dedifferentiated Proximal Tubules in Cystic Kidney Disease

Ectopic cAMP signaling is pathologic in polycystic kidney disease; however, its spatiotemporal actions are unclear. We characterized the expression of phosphorylated Creb (p-Creb), a target and mediator of cAMP signaling, in developing and cystic kidney models. We also examined tubule-specific effects of cAMP analogs in cystogenesis in embryonic kidney explants. In wild-type mice, p-Creb marked nephron progenitors (NP), early epithelial NP derivatives, ureteric bud, and cortical stroma; p-Creb was present in differentiated thick ascending limb of Henle, collecting duct, and stroma; however, it disappeared in mature NP-derived proximal tubules. In Six2cre;Frs2αFl/Fl mice, a renal cystic model, ectopic p-Creb stained proximal tubule-derived cystic segments that lost the differentiation marker lotus tetragonolobus lectin. Furthermore, lotus tetragonolobus lectin-negative/p-Creb-positive cyst segments (re)-expressed Ncam1, Pax2, and Sox9 markers of immature nephron structures and dedifferentiated proximal tubules after acute kidney injury. These dedifferentiation markers were co-expressed with p-Creb in renal cysts in Itf88 knockout mice subjected to ischemia and Six2cre;Pkd1Fl/Fl mice, other renal cystogenesis models. 8-Br-cAMP addition to wild-type embryonic kidney explants induced proximal tubular cystogenesis and p-Creb expression; these effects were blocked by co-addition of protein kinase A inhibitor. Thus p-Creb/cAMP signaling is appropriate in NP and early nephron derivatives, but disappears in mature proximal tubules. Moreover, ectopic p-Creb expression/cAMP signaling marks dedifferentiated proximal tubular cystic segments. Furthermore, proximal tubules are predisposed to become cystic after cAMP stimulation.