Phosphorylation and consequent stimulation of the tyrosine kinase c-Abl by PKA in mouse spermatozoa; its implications during capacitation

Upon ejaculation, spermatozoa undergo a series of post-translational modifications in a process known as capacitation in order to prepare for fertilization. In the absence of capacitation, fertilization cannot occur. Spermatozoa are unusual in that one of the hallmarks of capacitation is a global up-regulation in phosphotyrosine expression, which is known to be mediated upstream by PKA. Little is known about the signaling events downstream of PKA apart from the involvement of SRC, as a key mediator of PKA-induced tyrosine phosphorylation in the sperm tail. Here we describe the presence of c-Abl in mouse spermatozoa. In vitro analysis confirmed that PKA can up-regulate c-Abl kinase activity. In vivo, this tyrosine kinase was found to associate, and become threonine phosphorylated by PKA in the sperm flagellum. By treating spermatozoa with hemolysin we could demonstrate that a significant proportion of the tyrosine phosphorylation associated with capacitation could be suppressed by the c-Abl inhibitor, Gleevac. This is the first report of c-Abl being up-regulated by PKA for any cell type. We present a model, whereby these kinases may operate together with SRC to ensure optimal levels of tyrosine phosphorylation in the sperm flagellum during the attainment of a capacitated state.     

Dissecting the regions of virion-associated Kaposi's sarcoma-associated herpesvirus complement control protein required for complement regulation and cell binding

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

The enzymes of the transsulfuration pathways: active-site characterizations

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Characterization of the side-chain hydroxyl moieties of residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity in E. coli cystathionine β-lyase

Cystathionine β-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of site-directed variants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity. The effect of these conservative substitutions on the k(cat)/K(m)(L-Cth) for the α,β-elimination of L-Cth ranges from a change of only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine replacement variant of S339. A novel role for residue S339 as a determinant of reaction specificity, via tethering of the catalytic base, K210, is demonstrated. Comparison of the kinetic parameters for L-Cth hydrolysis with those for the inhibition of eCBL by aminoethoxyvinylglycine (AVG) indicates that Y238 interacts with the distal carboxylate group of the substrate. The 22 and 50-fold increases in the K(m)(L-Cth) and K(i)(AVG) resulting from replacement of Y56 with phenylalanine suggest that this residue may interact with the distal amino group of these compounds, although an indirect role in binding is more likely. The near-native k(cat)/K(m)(L-Cth) and pH profile of the eCBL-Y111F variant demonstrate that residue Y111 does not play a role in proton transfer. The understanding of the eCBL active site and of the determinants of substrate and reaction specificity resulting from this work will facilitate the design of inhibitors, as antibacterial therapeutics, and the engineering of enzymes dependent on the catalytically versatile pyridoxal 5'-phosphate cofactor to modify reaction specificity.     

The clinical significance,natural history and predictors of bone marrow lesion change over eight years

Introduction

There is increasing evidence to suggest that bone marrow lesions (BMLs) play a key role in the pathogenesis of osteoarthritis (OA). However, there is a lack of long term data. The aim of this study was to describe the natural history of knee BMLs, their association with knee pain and examine predictors of BML change over eight years.

Methods

A total of 198 subjects (109 adult offspring of subjects who had a knee replacement and 89 community-based controls) were studied. Knee pain and BML size were assessed at two and ten year visits.

Results

At the two year visit, 64% of participants (n = 127) had 229 BMLs (34% patella, 26% femoral and 40% tibial). Over eight years, 24% (55/229) increased in size, 55% (125/229) remained stable and 21% (49/229) decreased in size or resolved completely. Of the participants without BMLs at baseline, 52% (37/71) developed incident BMLs.After adjusting for confounders, eight year change in total BML size was associated with change in knee pain in offspring (β = 2.50, 95% confidence interval (CI) 0.96 to 4.05) but not controls. This association was stronger in males. Incident BMLs were associated with increase in pain (β = 3.60, 95% CI 1.14 to 6.05). Body mass index (BMI) and strenuous activity (but not radiographic osteoarthritis or smoking) were associated with an increase in BML size.

Conclusion

In this midlife cohort, the proportion of BMLs increasing in size was similar to those decreasing in size with the majority remaining stable. Change in BMLs was predicted by BMI and strenuous activity. An increase in BML size or a new BML resulted in an increase in pain especially in males and those with a family history of OA.     

Genotype Distribution,Viral Load and Clinical Characteristics of Infants with Postnatal or Congenital Cytomegalovirus Infection

Background

Congenital cytomegalovirus infection is a leading cause of long-term sequelae. Cytomegalovirus is also frequently transmitted to preterm infants postnatally, but these infections are mostly asymptomatic. A correlation between cytomegalovirus genotypes and clinical manifestations has been reported previously in infants with congenital infection, but not in preterm infants with postnatal infection.

Objectives

The main objective of this study was to investigate cytomegalovirus genotype distribution in postnatal and congenital cytomegalovirus infection and its association with disease severity.

Methods

Infants admitted to the neonatal intensive care unit of the University Medical Center Utrecht, The Netherlands between 2003–2010 and diagnosed with postnatal or congenital cytomegalovirus infection were included. Classification of cytomegalovirus isolates in genotypes was performed upon amplification and sequencing of the cytomegalovirus UL55 (gB) and UL144 genes. Clinical data, cerebral abnormalities, neurodevelopmental outcome and viral load were studied in relation to genotype distribution.

Results

Genotyping results were obtained from 58 preterm infants with postnatal cytomegalovirus infection and 13 infants with congenital cytomegalovirus infection. Postnatal disease was mild in all preterm infants and all had favourable outcome. Infants with congenital infection were significantly more severely affected than infants with postnatal infection. Seventy-seven percent of these infants were symptomatic at birth, 2/13 died and 3/13 developed long-term sequelae (median follow-up 6 (range 2–8) years). The distribution of cytomegalovirus genotypes was comparable for postnatal and congenital infection. UL55 genotype 1 and UL144 genotype 3 were predominant genotypes in both groups.

Conclusions

Distribution of UL55 and UL144 genotypes was similar in asymptomatic postnatal and severe congenital CMV infection suggesting that other factors rather than cytomegalovirus UL55 and UL144 genotype are responsible for the development of severe disease.