[Show abstract][Hide abstract] ABSTRACT: Calnexin (CANX) and calreticulin (CALR) chaperones mediate nascent glycoprotein folding in the endoplasmic reticulum. Here we report that these chaperones have distinct roles in male and female fertility. Canx null mice are growth retarded but fertile. Calr null mice die during embryonic development, rendering indeterminate any effect on reproduction. Therefore, we conditionally ablated Calr in male and female germ cells using Stra8 (mcKO) and Zp3 (fcKO) promoter-driven Cre recombinase, respectively. Calr mcKO male mice were fertile, but fcKO female mice were sterile despite normal mating behavior. Strikingly, we found that Calr fcKO female mice had impaired folliculogenesis and decreased ovulatory rates due to defective proliferation of cuboidal granulosa cells. Oocyte-derived, TGF-beta family proteins play a major role in follicular development and molecular analysis revealed that the normal processing of GDF9 and BMP15 was defective in Calr fcKO oocytes. These findings highlight the importance of CALR in female reproduction and demonstrate that compromised CALR function leads to ovarian insufficiency and female infertility.
[Show abstract][Hide abstract] ABSTRACT: A family of 10 new cyclometalated platinum(II) complexes have been prepared that incorporate a cyclometalating tridentate ligand together with a monodentate thiolate ligand. The complexes are phosphorescent in solution at room temperature. In comparison to the parent complexes containing a chloride coligand, the emission of the thiolate complexes is strongly red-shifted, increasingly so with the electron richness of the thiolate. Electrochemical data and TD-DFT calculations indicate that the change is due to the introduction of a charge-transfer state, but nitro-substituted thiolates behave differently.
[Show abstract][Hide abstract] ABSTRACT: The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide bonds. These are introduced into client proteins by ER resident oxidoreductases, including ER oxidoreductin 1 (Ero1). Ero1 is usually considered to function in a linear pathway, by 'donating' a disulfide bond to protein disulfide isomerase (PDI) and receiving electrons that are passed on to the terminal electron acceptor molecular oxygen. PDI engages with a range of clients as the direct catalyst of disulfide bond formation, isomerization or reduction. In this paper, we will consider the interactions of Ero1 with PDI family proteins and chaperones, highlighting the effect that redox flux has on Ero1 partnerships. In addition, we will discuss whether higher order protein complexes play a role in Ero1 function.
Philosophical Transactions of The Royal Society B Biological Sciences 05/2013; 368(1617):20110403. DOI:10.1098/rstb.2011.0403 · 7.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim:
Ero proteins are central to oxidative protein folding in the endoplasmic reticulum (ER), but their expression varies in a tissue-specific manner. The aim of this work was to establish the expression of Ero1α in the digestive system and to examine the behavior of Ero1α in premalignant Barrett's esophagus, esophageal (OE) and gastric cancers and esophageal cancer cell lines.
Ero1α is expressed in the columnar epithelium of Barrett's tissue, and in OE tumors and gastric tumors. Homocysteine, a precursor in the metabolism of cysteine and methionine, induces the active Ox1 form of Ero1α in the OE cancer cell line OE33.
These results demonstrate for the first time that Ero1α can sense the level of an amino acid precursor, identifying a potential link between diet, antioxidants, and oxidative protein folding in the ER.
The high expression of Ero1α in cancers of the esophagus and stomach demonstrates the importance of ER redox regulation in the gastro-intestinal (GI) tract in health and disease. Proteins and metabolites involved in disulfide bond formation and redox regulation may be suitable targets for both biomarker and drug development in GI cancer.
[Show abstract][Hide abstract] ABSTRACT: In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situations found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation.
Cold Spring Harbor perspectives in biology 06/2012; 4(8):a012872. DOI:10.1101/cshperspect.a012872 · 8.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for both sperm migration from the uterus into the oviduct and sperm primary binding to the zona pellucida (ZP). Here we show that the testis-specific protein disulfide isomerase homolog (PDILT) cooperates with the testis-specific calreticulin-like chaperone, calsperin (CALR3), in the endoplasmic reticulum and plays an indispensable role in the disulfide-bond formation and folding of ADAM3. Pdilt(-/-) mice were male infertile because ADAM3 could not be folded properly and transported to the sperm surface without the PDILT/CALR3 complex. Peculiarly we find that not only Pdilt(-/-), but also Adam3(-/-), spermatozoa effectively fertilize eggs when the eggs are surrounded in cumulus oophorus. These findings reveal that ADAM3 requires testis-specific private chaperones to be folded properly and that the principle role of ADAM3 is for sperm migration into the oviduct but not for the fertilization event. Moreover, the importance of primary sperm ZP binding, which has been thought to be a critical step in mammalian fertilization, should be reconsidered.
Proceedings of the National Academy of Sciences 03/2012; 109(10):3850-5. DOI:10.1073/pnas.1117963109 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein disulfide isomerase (PDI) and its homologs have essential roles in the oxidative folding and chaperone-mediated quality control of proteins in the secretory pathway. In this review, the importance of PDI in health and disease will be examined, using examples from the fields of lipid homeostasis, hemostasis, infectious disease, cancer, neurodegeneration, and infertility. RECENT ADVANCES: Recent structural studies, coupled with cell biological, biochemical, and clinical approaches, have demonstrated that PDI family proteins are involved in a wide range of physiological and disease processes.
Critical issues in the field include understanding how and why a PDI family member is involved in a given disease, and defining the physiological client specificity of the various PDI proteins when they are expressed in different tissues.
Future directions are likely to include the development of new and more specific reagents to study and manipulate PDI family function. The development of conditional mouse models in concert with clinical data will help us to understand the in vivo function of the different PDIs at the organism level. Taken together with advances in structural biology and biochemical studies, this should help us to further understand and modify PDIs' functional interactions.
[Show abstract][Hide abstract] ABSTRACT: Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.
[Show abstract][Hide abstract] ABSTRACT: Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate
to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific
counterparts of CANX and CALR, respectively. Here, we show that Calr3−/− males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct
and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation
of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly
required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific
CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification
and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception
and treatment of unexplained male infertility.
[Show abstract][Hide abstract] ABSTRACT: The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR,
-DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules
have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious
diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly
and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported
by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model
antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR
complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides
than other human MHC class II proteins and frequently present viral peptides.
[Show abstract][Hide abstract] ABSTRACT: Mammalian fertilization comprises sperm migration through the female reproductive tract, biochemical and morphological changes to sperm, and sperm-egg interaction in the oviduct. Recent gene knockout approaches in mice have revealed that many factors previously considered important for fertilization are largely dispensable, or if they are essential, they have an unexpected function. These results indicate that what has been observed in in vitro fertilization (IVF) differs significantly from what occurs during "physiological" fertilization. This Review focuses on the advantages of studying fertilization using gene-manipulated animals and highlights an emerging molecular mechanism of mammalian fertilization.
The Journal of clinical investigation 04/2010; 120(4):984-94. DOI:10.1172/JCI41585 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum (ER) plays a critical role as a compartment for protein folding in eukaryotic cells. Defects in protein folding contribute to a growing list of diseases, and advances in our understanding of the molecular details of protein folding are helping to provide more efficient ways of producing recombinant proteins for industrial and medicinal use. Moreover, research performed in recent years has shown the importance of the ER as a signalling compartment that contributes to overall cellular homeostasis. Hamlet's castle provided a stunning backdrop for the latest European network meeting to discuss this subject matter in Elsinore, Denmark, from 3 to 5 June 2009. Organized by researchers at the Department of Biology, University of Copenhagen, the meeting featured 20 talks by both established names and younger scientists, focusing on topics such as oxidative protein folding and maturation (in particular in the ER, but also in other compartments), cellular redox regulation, ER-associated degradation, and the unfolded protein response. Exciting new advances were presented, and the intimate setting with about 50 participants provided an excellent opportunity to discuss current key questions in the field.
[Show abstract][Hide abstract] ABSTRACT: Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Delta-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.
Molecular Biology of the Cell 09/2007; 18(8):2795-804. DOI:10.1091/mbc.E07-02-0147 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Misfolding of major histocompatibility complex (MHC) class I molecules has been implicated in the rheumatic autoimmune disease ankylosing spondylitis (AS), and has been linked to the unfolded protein response (UPR) in rodent AS models. XBP1 and ATF6alpha are two important transcription factors that initiate and co-ordinate the UPR. Here we show that misoxidised MHC class I heavy chains activate XBP1 processing in a similar manner to tunicamycin, with tunicamycin and dithiothreitol (DTT) inducing differential XBP1 processing. Unexpectedly, ATF6alpha mRNA is alternatively spliced during reducing stress in lymphocytes. This shorter ATF6alpha message lacks exon 7 and may have a regulatory role in the UPR.
[Show abstract][Hide abstract] ABSTRACT: Increasing enrichment of dimethyl sulfoxide, DMSO, in DMSO-water mixtures causes a reversal in the thermodynamic dissociation
as, and has a marked effect on the redox potentails of the thiolic and amino groups in cysteine and the protein disulfide isomerase
(PDI) mimic BMC, Vectrase. This paper illustrates the effect of a hydrogen-bonding environment on the ionisation and redox
properties of thiol groups in amino acids. A combination of potentiometry and Raman spectroscopy was applied to rationalise
the observations. Intracellular environments are full of hydrophobic, hydrogen-bonding environments. The results illustrate
the profound effects of the local environment on the thiol group.