The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.
To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.
- MeSH
- Transcription, Genetic physiology MeSH
- Genome physiology MeSH
- Hemoglobins genetics metabolism MeSH
- Cathepsin D genetics metabolism MeSH
- Ixodes enzymology genetics MeSH
- Protein Processing, Post-Translational physiology MeSH
- Arthropod Proteins genetics metabolism MeSH
- Recombinant Proteins genetics metabolism MeSH
- Intestines enzymology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The slowly metabolized proteins of the extracellular matrix, typically collagen and elastin, accumulate reactive metabolites through uncontrolled non-enzymatic reactions such as glycation or the products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups). A typical example of these non-enzymatic changes is the formation of advanced glycation end-products (AGEs), resulting from the reaction of carbohydrates with the free amino group of proteins. The accumulation of AGEs and the resulting structural alterations cause altered tissue properties (increased stiffness, reduced elasticity) that contribute to their reduced catabolism and to their aging. Posttranslational nonenzymatic modifications of the proteins of the extracellular matrix (the formation of a typical AGE product - pentosidine) were studied in three types of tissue of three rat strains subjected to a high-fructose diet. Chronic (three-week) hyperglycemia (resulting from fructose loading) caused a significant increase in pentosidine concentration mainly in the aorta and skin of the three rat strains (Lewis, Wistar and hereditary hypertriglyceridemic rats).
- MeSH
- Aorta metabolism MeSH
- Arginine analogs & derivatives metabolism MeSH
- Endothelium, Vascular metabolism MeSH
- Dietary Carbohydrates metabolism MeSH
- Species Specificity MeSH
- Extracellular Matrix metabolism MeSH
- Fructose metabolism MeSH
- Hyperglycemia complications metabolism MeSH
- Hypertriglyceridemia complications metabolism MeSH
- Rats MeSH
- Skin metabolism MeSH
- Lysine analogs & derivatives metabolism MeSH
- Statistics, Nonparametric MeSH
- Protein Processing, Post-Translational physiology MeSH
- Rats, Inbred Lew MeSH
- Rats, Wistar MeSH
- Glycation End Products, Advanced metabolism MeSH
- Tendons metabolism MeSH
- Tissue Distribution MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.
- MeSH
- Ataxia Telangiectasia Mutated Proteins MeSH
- DNA-Binding Proteins metabolism MeSH
- DNA-(Apurinic or Apyrimidinic Site) Lyase MeSH
- DNA Breaks, Double-Stranded * MeSH
- Phosphoproteins genetics metabolism MeSH
- Phosphorylation MeSH
- DNA Breaks, Single-Stranded * MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Genomic Instability * physiology MeSH
- DNA Repair * physiology MeSH
- Protein Processing, Post-Translational physiology MeSH
- Protein Serine-Threonine Kinases metabolism MeSH
- Cell Cycle Proteins metabolism MeSH
- Protein Structure, Tertiary genetics MeSH
- Zinc Fingers genetics MeSH
- Check Tag
- Humans MeSH
The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.
- MeSH
- Adaptor Proteins, Signal Transducing genetics metabolism MeSH
- Cell Membrane * metabolism MeSH
- Endocytosis MeSH
- Phosphoric Monoester Hydrolases genetics metabolism MeSH
- Phosphorylation drug effects MeSH
- HeLa Cells MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Humans MeSH
- Mutation, Missense MeSH
- Multiprotein Complexes genetics metabolism MeSH
- Protein Processing, Post-Translational physiology drug effects MeSH
- GTPase-Activating Proteins genetics metabolism MeSH
- rab5 GTP-Binding Proteins genetics metabolism MeSH
- Tungsten Compounds pharmacology MeSH
- Amino Acid Substitution MeSH
- Substrate Specificity genetics drug effects MeSH
- Protein Binding genetics drug effects MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
Studie se zabývá posttranslačními modifikacemi proteinu – glykací tkáně intervertebrální ploténky a stanovením jednoho z pokročilých koncových produktů – pentosidinu ve vztahu k věku. Pentosidin byl detekován v hydrolyzátu meziobratlových plotének u osob ve věku mezi 16 a 95 lety. Sto čtyřicet dva vzorků bylo analyzováno vysoce citlivou kapalinovou chromatografií a zjištěné množství pentosidinu bylo statisticky vyhodnoceno. Koeficient korelace závislosti množství pentosidinu na věku je r = 0,92. Výsledky práce potvrzují skutečnost, že je možné použít stanovení obsahu pentosidinu v tkáni meziobratlové ploténky k přibližnému stanovení věku. Nicméně další experimenty by měly být provedeny za rozdílných podmínek posmrtného rozkladu.
The study deals with the post-translational modifications of proteins – glycation of the tissue of the intervertebral disc and determination of one of advanced glycation end's products – pentosidine in the relation to the age. Pentosidine was detected in the hydrolysate of the intervertebral discs from persons between the ages of 16 and 95 years. 142 samples were analysed by high performance liquid chromatography, and the detected amounts of pentosidine were processed statistically. The coefficient of correlation of dependence of the amount of pentosidine on the age amounts to r = 0.92. The results of the work testify to the fact that it is possible to use the detection of pentosidine in the tissue of the intervertebral disc for the estimation of the age. Nevertheless subsequent experiments should be done under different conditions post-mortem decomposition.
- MeSH
- Arginine analogs & derivatives isolation & purification MeSH
- Research Support as Topic MeSH
- Glycosylation drug effects MeSH
- Humans MeSH
- Intervertebral Disc chemistry MeSH
- Cadaver MeSH
- Protein Processing, Post-Translational physiology MeSH
- Forensic Medicine methods MeSH
- Age Determination by Skeleton methods MeSH
- Chromatography, High Pressure Liquid methods utilization MeSH
- Check Tag
- Humans MeSH
