kDa Precursor Research Articles

Construction of the Arabidopsis isogenic lines containing dually localized protein TROL only in the inner chloroplast envelope membrane

The thylakoid rhodanese-like protein (TROL) is located at the end of the photosynthetic electron transport chain, at the vicinity of photosystem I, where it dynamically interacts with the ferredoxin:NADP+ oxidoreductase (FNR) and is postulated to facilitate the transfer of electrons from reduced ferredoxin (Fd) to NADP+. TROL is one of the few so far known dually localized chloroplast proteins. Besides being localized in the thylakoid membranes as the 66 kDa mature form, it has also been found in the inner envelope membrane of chloroplasts as the 70 kDa precursor. In thylakoids, the interaction between TROL and FNR acts like a switch, prioritizing the photosynthetic electron destination sinks according to the organellar needs. The role of TROL in the chloroplast inner envelope membrane is, however, presently unknown. By engineering the presequence protease processing site, a single amino acid exchange of Ala67 to Ile67 has been introduced to TROL, leading to inhibited cleavage of the presequence and resulting in protein incorporation at the inner envelope membrane. In this work, we engineered the Arabidopsis mutant plants that contain TROL almost exclusively in the inner envelope membrane (TROL-IE). To facilitate studying the role of this protein in this chloroplast compartment, we also produced the antiserum specific for the IE form of the TROL.

The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor.

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and β chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand—hepatocyte growth factor (HGF)—such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.

Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αβ+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3−CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αβ+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αβ+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

Role of testicular interleukin-1alpha tIL-1alpha in testicular physiology and disease.

This review focuses on the role of the cytokine interleukin-1alpha (IL-1alpha) in the testis; elaborating upon its importance during the complex process of spermatogenesis while relating this cytokine to some of the pathophysiological states affecting the testis. IL-1alpha, a proinflammatory cytokine, is expressed constitutively by the intact adult rat testis where it acts on germ, Sertoli and Leydig cells to regulate germ cell proliferation and steroidogenesis. The sequence identity of testicular IL-1alpha matches with the one secreted by activated macrophages in systemic immunity. The classical macrophage IL-1alpha is produced as 32 kDa precursor protein which is processed to mature 17 kDa IL-1alpha and a 16 kDa propiece. The rat testicular IL-1alpha, mainly secreted by Sertoli cells, was found to have molecular heterogeneity that can be observed both at the transcriptional and the translational levels. In the rat testis, two transcripts were found to be expressed with 941 bp and 767 bp (that lacks 174 bp) which were translated into 32 kDa and 24 kDa precursor proteins, respectively. The 32 kDa precursor protein is processed to the 17 kDa mature IL-1alpha. Identical transcripts are also shown to be present in cat, dog and pig. Most of the functional role is assigned to the mature 17 kDa IL-1alpha isoform. However, functional analysis of recombinant rat IL-1alpha isoforms showed that there was a clear biopotency difference between these forms in order of 17 kDa IL-1alpha>32proIL-1alpha>24proIL-1alpha. Furthermore, the mature 17 kDa tIL-1alpha has also been implicated in pathologies such as orchitis, relapse of acute lymphoblastic leukemia (ALL) in the testis and infertility disorders in men. Thus, tIL-1alpha may play an important functional role both in coordination of normal testicular physiology as well as in contributing to the disease states in the testis.

A dock derived compound against laminin receptor (37 LR) exhibits anti-cancer properties in a prostate cancer cell line model.

Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor (37 LR). 37/67 LR is a strong clinical correlate for progression, aggression, and chemotherapeutic relapse of several cancers including breast, prostate, and colon. The ability of 37/67 LR to promote cancer cell aggressiveness is further increased by its ability to transduce physiochemical and mechanosensing signals in endothelial cells and modulate angiogenesis. Recently, it was demonstrated that 37/67 LR modulates the anti-angiogenic potential of the secreted glycoprotein pigment epithelium-derived factor (PEDF). Restoration of PEDF balance is a desirable therapeutic outcome, and we sought to identify a small molecule that could recapitulate known signaling properties of PEDF but without the additional complications of peptide formulation or gene delivery safety validation. We used an in silico drug discovery approach to target the interaction interface between PEDF and 37 LR. Following cell based counter screening and binding validation, we characterized a hit compound’s anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis.

Crocodylian nuclear factor kappa B.

We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element.

Mutations affecting the transmembrane domain of the LDL receptor: impact of charged residues on the membrane insertion.

Familial hypercholesterolemia (FH) is caused by mutations in the low density lipoprotein receptor (LDLR) gene. To study the impact of mutations affecting the hydrophobic transmembrane domain of the LDLR, each of the 22 amino acids of the transmembrane domain was individually mutated to arginine. The more centrally in the transmembrane domain an arginine was located, the lower amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum were observed. This led to lower amounts of the 160 kDa mature LDLR on the cell surface. For the mutants V797R-LDLR, L798R-LDLR and L799R-LDLR a proportion of full-length receptors including the transmembrane and cytoplasmic domains, was secreted into the endoplasmic reticulum lumen to appear in the culture medium. When the transmembrane domain of the epidermal growth factor receptor (EGFR) was replaced by that of the mutant L799R-LDLR, similar effects were observed for the EGFR as for L799R-LDLR. Introducing arginines in the transmembrane domain of the LDLR also affected metalloproteinase cleavage of the ectodomain and γ-secretase cleavage within the transmembrane domain. The most likely explanation for the low amounts of the 120 kDa precursor is that a basic residue in the hydrophobic transmembrane domain prevents the mutant LDLR from being inserted in the endoplasmic reticulum membrane from the Sec61 translocon complex. As a consequence, quality control systems could be activated. However, our data indicate that proteasomal degradation, lysosomal degradation, autophagy or ectodomain cleavage were not the underlying mechanism for degradation of these mutant LDLRs.

Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle’s integrin β1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle’s membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

The Transition of the 37-Kda Laminin Receptor (Rpsa) to Higher Molecular Weight Species: Sumoylation or Artifact?

The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

Abstract 3257: ERC/mesothelin is expressed in human gastric cancer tissues and cell lines

Abstract Background: ERC/mesothelin is expressed in mesothelioma and other malignancies. The ERC/mesothelin gene (MSLN) encodes a 71 kDa precursor protein, which is cleaved to yield 31 kDa N-terminal (N-ERC/mesothelin) and 40 kDa C-terminal (C-ERC/mesothelin) proteins. N-ERC/mesothelin is a soluble protein and has been reported to be a diagnostic serum marker of mesothelioma and ovarian cancer. Gastric cancer tissue also expresses C-ERC/mesothelin, but the significance of serum N-ERC levels for diagnosing gastric cancer has not yet been studied. We examined the latter issue in this study as well as C-ERC/mesothelin expression in human gastric cancer tissues and cell lines. Methods: We immunohistochemically examined C-ERC/mesothelin expression in tissue samples from 50 cases of gastric cancer, and we also assessed the C-ERC/mesothelin expression of 6 cancer cell lines (MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1) using the reverse transcription-polymerase chain reaction, flow cytometry, immunohistochemistry, and immunoblotting. We also examined the N-ERC/mesothelin concentrations of the supernatants of cultured cells and the sera of gastric cancer patients using an ELISA. Results: N-ERC/mesothelin was detected in the supernatants of 3 cell lines (MKN-1, NUGC-4, and TMK-1) by ELISA, but its concentration in the sera of gastric cancer patients was almost same as that observed in the sera of the normal controls. In the gastric cancer tissues, C-ERC/mesothelin expression was associated with lymphatic invasion, as reported by Einama et al. Conclusion: N-ERC/mesothelin was secreted into the supernatants of gastric cancer cell lines, but did not appear to be a useful serum marker of gastric cancer. Citation Format: Tomoaki Ito, Kazunori Kajino, Masaaki Abe, Koichi Sato, Hiroshi Maekawa, Mutsumi Sakurada, Hajime Orita, Ryo Wada, Yoshiaki Kajiyama, Okio Hino. ERC/mesothelin is expressed in human gastric cancer tissues and cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3257. doi:10.1158/1538-7445.AM2014-3257

Megakaryocytic potentiating factor and mature mesothelin stimulate the growth of a lung cancer cell line in the peritoneal cavity of mice.

The mesothelin (MSLN) gene encodes a 71 kilodalton (kDa) precursor protein that is processed into megakaryocytic potentiating factor (MPF), a 31 kDa protein that is secreted from the cell, and mature mesothelin (mMSLN), a 40 kDa cell surface protein. The mMSLN binds to CA125, an interaction that has been implicated in the intra-cavitary spread of mesothelioma and ovarian cancer. To better define the role of MPF and mMSLN, growth of the lung cancer cell line A549 was evaluated in immuno-deficient mice with inactivation of the Msln gene. We observed that Msln–/– mice xenografted with intraperitoneal A549 tumors survive significantly long than tumor-bearing Msln+/+ mice. When tumor-bearing Msln–/– mice are supplemented with recombinant MPF (and to a lesser extent mMSLN), most of this survival advantage is lost. These studies demonstrate that MPF and mMSLN have an important role in the growth of lung cancer cells in vivo and raise the possibility that inactivation of MPF may be a useful treatment for lung and other MSLN expressing cancers.

The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a ∼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of ∼24 kDa with pI values of ∼6.7, 6.9, and 7.0 and two spots of ∼22 and ∼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.

Perturbations to the Ubiquitin Conjugate Proteome in Yeast Δubx Mutants Identify Ubx2 as a Regulator of Membrane Lipid Composition

Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested. Here, we use mass spectrometry to identify ubiquitin conjugates that accumulate in cdc48 and ubx mutants. Different ubx mutants exhibit unique patterns of conjugate accumulation that point to functional specialization of individual Ubx proteins. To validate our findings, we examined in detail the endoplasmic reticulum-bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. Mutant ubx2Δ cells are deficient in both cleaving the ubiquitinated 120 kDa precursor of Spt23 to form active p90 and in localizing p90 to the nucleus, resulting in reduced expression of the target gene OLE1, which encodes fatty acid desaturase. Our findings provide a resource for future investigations on Cdc48, illustrate the utility of proteomics to identify ligands for specific ubiquitin receptor pathways, and uncover Ubx2 as a key player in the regulation of membrane lipid biosynthesis.

Development and Characterization of Six Monoclonal Antibodies to Hemagglutinin-70 ofClostridium botulinumand Their Application in a Sandwich ELISA

Botulinum neurotoxins (BoNT) are produced by Clostridium botulinum and cause severe neuroparalytic disease that if not treated quickly is often fatal. The toxin is produced as a 150 kDa precursor protein (holotoxin) that is enzymatically cleaved to form two subunits, heavy and light chains, linked by a single disulfide bond. Seven toxin serotypes are known. BoNT serotypes A1 and B1 are secreted as precursor toxic complexes (PTC) containing of the toxin and non-toxic associated proteins (NAPs) consisting of non-toxic hemagglutinin proteins (HA), designated HA17, HA34, and HA70, and a 120 kDa non-toxin non-hemagglutinin (NTNH) protein. The exact contribution of the NAPs in disease is not known, but it is thought that they protect the toxin as it passes through the harsh environment of the stomach. The structure of the complex is also poorly understood, although recent models suggest that for each molecule of toxin the PTC contains one molecule of the NTNH and multiple copies of each HA. In this paper we describe six monoclonal antibodies that specifically bind the HA70 protein found in the PTC of BoNT/A1 and /B1. Based on these antibodies, we demonstrate a rapid sandwich ELISA assay for detecting HA70.

Genetic structure and expression of the surface glycoprotein GP82, the main adhesin of Trypanosoma cruzi metacyclic trypomastigotes.

T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. The metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. The function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3′UTR.

Caspase-1 and IL-1β Processing in a Teleost Fish

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β.

Autoproteolytic and Catalytic Mechanisms for the β-Aminopeptidase BapA—A Member of the Ntn Hydrolase Family

The β-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal β-amino acid residues from peptides. We determined the crystal structures of the native (αβ)₄ heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.

Alteration in the processing of the ACRBP/sp32 protein and sperm head/acrosome malformations in proprotein convertase 4 (PCSK4) null mice

Proprotein convertase 4 (PCSK4) is a member of a family of proprotein convertases that convert inactive precursor proteins into their mature and active forms. PCSK4 is expressed by testicular germ cells and localizes to the sperm acrosome, suggesting roles in fertilization. Mice lacking PCSK4 exhibit a profound fertility defect; yet, to date, few substrates for PCSK4 are known. In this study, two-dimensional differential in-gel electrophoresis analysis was carried out in order to identify proteins that are altered in spermatozoa from PCSK4 null mice. Herein, we report that the sperm fertilization molecule acrosin-binding protein (ACRBP)/sp32, which normally undergoes processing from a 58.5 kDa precursor to a 27.5 kDa mature form, is not proteolytically processed in PCSK4 null mice and thus may be a substrate for PCSK4. However, analysis of the ACRBP sequence did not show a strong consensus site for convertase cleavage, suggesting that ACRBP processing may require the activity of a yet unknown enzyme that itself may be a PCSK4 substrate. Further analysis of spermatozoa from the PCSK4 null mice showed that proacrosin did not undergo autoactivation, supporting a role for the mature form of ACRBP in the regulation of proacrosin conversion into different acrosin isoforms. Finally, examination of ACRBP localization revealed a previously undetected morphological defect in the head/acrosomes of spermatozoa from PCSK4 null mice. Taken together, these results demonstrate that the fertility defect in the PCSK4 null mice may in part be due to altered ACRBP protein processing as well as abnormalities in the sperm head/acrosome.

How Does Oxidative Stress Affect Inflammasome Activation?

The innate immune system activated as a first line of response to host defense during bacterial infections and injury. It plays a key role in the initial recognition followed by the production of pro-inflammatory cytokines and chemokines to halt invading pathogens [1]. The innate immune response relies on recognition of evolutionarily conserved structures of pathogens, known as pathogen-associated molecular patterns (PAMPs), through a limited number of germ line-encoded pattern recognition receptors (PRRs) [2]. Among these, the family of Toll-like receptors (TLRs) has been studied most extensively for immune response [3]. Bacterial cell wall components such as lipopolysaccharide (LPS) are the most potent PAMPs known and responsible for the inflammatory response observed during bacterial infections. PAMPs trigger innate immune response by producing the major inflammatory cytokines such as IL-1b and IL-18 which by autocrine and paracrine manner generates a number of other cytokines leading to chronic inflammation, profound vasodilatation, organ failure and dysfunction [4-7]. IL-1b and IL-18 are essential for host defense, innate immune response, and mediate the pathogenesis of many inflammatory diseases such as sepsis [8]. Generally, cytokines such as IL-1b, IL-18 and IL-33, lack a signal peptide for the classic endoplasmic reticulum-mediated release of proteins by exocytosis, are synthesized as an inactive precursor by NF-ĸB-mediated signalosome [9]. In order to be activated, these cytokines need to be cleaved by an intracellular cysteine protease called caspase-1. For example, activation of caspase-1 cleaves 31 kDa precursor form of IL-1b into a bioactive 17kDa form [10]. However, caspase-1 is itself synthesized as a zymogen which needs to be processed in to 20 and 10 kDa subunits to form an active enzyme. Activation of caspase-1 is mediated by recently discovered multi-protein complex called inflammasome. In response to microbial stimuli or injury, a major NOD like receptor (NLR) family member assembles in a large multi-protein complex in association with ASC and pro-caspase-1 leading to autocatalytic activation of caspase-1 [11]. NLR family proteins such as NALP1, NALP3 and NALP4 (also called NLRP1, NLRP2 and NLRP4) and a cytoplasmic protein AIM2 (absent in melanoma 2; which does not belong to NLR family) recruit CARD domain containing protein called PYCARD (or ASC) and pro-caspase-1 to from active inflammasome for the processing of inflammatory cytokines [11]. Out of these known inflammasomes, NALP3 inflammasome is extensively investigated and found to be involved in a number of inflammatory pathologies. In fact, recent emerging studies indicate that inhibition of NALP3 inflammasome -mediated innate immune response could be a novel therapeutic strategy for the prevention of inflammatory complications such as sepsis, arthritis, Crohn’s disease, Alzheimer’s, gout and atherosclerosis [12,13].