scholarly journals Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

2016 ◽  
Vol 113 (26) ◽  
pp. 7142-7147 ◽  
Author(s):  
Rutger A. F. Gjaltema ◽  
Miesje M. van der Stoel ◽  
Miriam Boersema ◽  
Ruud A. Bank
Keyword(s):  
Posttranslational Modifications ◽  
Molecular Level ◽  
Splice Variants ◽  
Active Form ◽  
Connective Tissue Disorder ◽  
Cross Linking ◽  
Ppiase Activity ◽  
Lysyl Hydroxylase ◽  
Bruck Syndrome ◽  
Lysine Hydroxylation

Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

Effects of elevated circulating IGF-1 on the extracellular matrix in "high-growth" C57BL/6J mice

1996 ◽  
Vol 271 (3) ◽  
pp. R696-R703 ◽  
Author(s):  
K. Reiser ◽  
P. Summers ◽  
J. F. Medrano ◽  
R. Rucker ◽  
J. Last ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulation Tissue ◽  
Posttranslational Modifications ◽  
Lysyl Oxidase ◽  
High Growth ◽  
Cross Linking ◽  
Type I ◽  
Igf Binding Proteins ◽  
Lysyl Hydroxylase ◽  
Collagen Cross Linking

Collagen biosynthesis was analyzed in C57BL/6J mice homozygous for the high-growth locus. Plasma levels of insulin-like growth factor-1 (IGF-1) were significantly elevated in high-growth mice at all ages studied (3 wk-6 mo); IGF-binding proteins were also elevated. Skin biopsies were obtained from mice aged 3, 6, and 9 wk under halothane anesthesia. Mice were killed at 6 mo of age. Collagen, expressed per weight of tissue, was significantly increased in all tissues from high-growth mice, as was collagen cross-linking, expressed as moles of cross-link per mole of collagen. Expression of types I and III collagen, lysyl oxidase, and lysyl hydroxylase was increased in all tissues analyzed. There was a preferential increase in type III expression relative to type I expression. Rate and extent of accumulation of collagen in granulation tissue were measured in polyvinyl alcohol sponges implanted subcutaneously; collagen accumulation was significantly greater in the high-growth mice. These results suggest that 1) elevated circulating IGF-1 may increase collagen deposition both in normal tissue as well as in granulation tissue by increasing collagen gene expression, 2) IGF-1 may increase collagen cross-linking by stimulating expression of lysyl oxidase, and 3) the preferential increase in dihydroxylated cross-links observed in high-growth mice may be due to the stimulation of lysyl hydroxylase expression by IGF-1. In summary, elevated levels of IGF-1 appear to affect collagen both quantitatively and qualitatively, primarily through their effects on gene expression of collagen and of those enzymes responsible for posttranslational modifications of collagen.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

Cysteine-Based Redox Sensing and Its Role in Signaling by Cyclic Nucleotide–Dependent Kinases in the Cardiovascular System

2019 ◽  
Vol 81 (1) ◽  
pp. 63-87 ◽  
Author(s):  
Friederike Cuello ◽  
Philip Eaton
Keyword(s):  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Level ◽  
Protein Kinase G ◽  
Cardiovascular Physiology ◽  
Homeostatic Regulation ◽  
Major Mechanism ◽  
Health And Disease ◽  
Redox Sensing ◽  
Cysteine Thiols

Oxidant molecules are produced in biological systems and historically have been considered causal mediators of damage and disease. While oxidants may contribute to the pathogenesis of disease, evidence continues to emerge that shows these species also play important regulatory roles in health. A major mechanism of oxidant sensing and signaling involves their reaction with reactive cysteine thiols within proteins, inducing oxidative posttranslational modifications that can couple to altered function to enable homeostatic regulation. Protein kinase A and protein kinase G are regulated by oxidants in this way, and this review focuses on our molecular-level understanding of these events and their role in regulating cardiovascular physiology during health and disease.

scholarly journals The Size of the Human Proteome: The Width and Depth

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Elena A. Ponomarenko ◽  
Ekaterina V. Poverennaya ◽  
Ekaterina V. Ilgisonis ◽  
Mikhail A. Pyatnitskiy ◽  
Arthur T. Kopylov ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Splice Variants ◽  
Meta Analysis ◽  
Human Proteome ◽  
Single Amino Acid ◽  
Analytical Sensitivity ◽  
Protein Coding ◽  
Single Chromosome ◽  
Independent Events ◽  
Specific Tissue

This work discusses bioinformatics and experimental approaches to explore the human proteome, a constellation of proteins expressed in different tissues and organs. As the human proteome is not a static entity, it seems necessary to estimate the number of different protein species (proteoforms) and measure the number of copies of the same protein in a specific tissue. Here, meta-analysis of neXtProt knowledge base is proposed for theoretical prediction of the number of different proteoforms that arise from alternative splicing (AS), single amino acid polymorphisms (SAPs), and posttranslational modifications (PTMs). Three possible cases are considered:(1)PTMs and SAPs appear exclusively in the canonical sequences of proteins, but not in splice variants;(2)PTMs and SAPs can occur in both proteins encoded by canonical sequences and in splice variants;(3)all modification types (AS, SAP, and PTM) occur as independent events. Experimental validation of proteoforms is limited by the analytical sensitivity of proteomic technology. A bell-shaped distribution histogram was generated for proteins encoded by a single chromosome, with the estimation of copy numbers in plasma, liver, and HepG2 cell line. The proposed metabioinformatics approaches can be used for estimation of the number of different proteoforms for any group of protein-coding genes.

Collagen cross-linking mediated by lysyl hydroxylase 2: an enzymatic battlefield to combat fibrosis

2019 ◽  
Vol 63 (3) ◽  
pp. 377-387 ◽  
Author(s):  
Bram Piersma ◽  
Ruud A. Bank
Keyword(s):  
Organ Failure ◽  
Molecular Basis ◽  
Repair Process ◽  
Cross Linking ◽  
Collagen Network ◽  
Lysyl Hydroxylase ◽  
Cross Links ◽  
Collagen Cross Linking ◽  
Excessive Accumulation

Abstract The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.

Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

2019 ◽  
Author(s):  
Michael Bæk, ◽  
Pablo Martín-Gago ◽  
Jonas S. Laursen ◽  
Julie L. H. Madsen ◽  
Saswati Chakladar ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Splice Variants ◽  
Peptide Sequence ◽  
Fluorescence Measurements ◽  
Lysine Residues ◽  
Covalent Adducts ◽  
Endogenous Proteins ◽  
Activity Dependent ◽  
Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-<i>N</i>-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-<i>N</i>-glutaryllysine and epsilon<i>-N</i>-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

2019 ◽  
Author(s):  
Michael Bæk, ◽  
Pablo Martín-Gago ◽  
Jonas S. Laursen ◽  
Julie L. H. Madsen ◽  
Saswati Chakladar ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Splice Variants ◽  
Peptide Sequence ◽  
Fluorescence Measurements ◽  
Lysine Residues ◽  
Covalent Adducts ◽  
Endogenous Proteins ◽  
Activity Dependent ◽  
Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-<i>N</i>-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-<i>N</i>-glutaryllysine and epsilon<i>-N</i>-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

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