scholarly journals SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

2007 ◽  
Vol 403 (2) ◽  
pp. 305-312 ◽  
Author(s):  
Alessandro Fraldi ◽  
Alessandra Biffi ◽  
Alessia Lombardi ◽  
Ilaria Visigalli ◽  
Stefano Pepe ◽  
...  
Keyword(s):  
Cysteine Residue ◽  
Chondrodysplasia Punctata ◽  
Post Translational Modification ◽  
Adeno Associated Virus ◽  
Diverse Range ◽  
Enhancing Effect ◽  
Multiple Sulfatase Deficiency ◽  
Sulfatase Activity ◽  
Lentivirus Vectors

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.

scholarly journals Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

2021 ◽  
Author(s):  
◽  
Jack Alexander Sissons
Keyword(s):  
Single Gene ◽  
Specific Protein ◽  
Rapid Identification ◽  
Phosphopantetheinyl Transferase ◽  
Post Translational Modification ◽  
Diverse Range ◽  
Peptide Motifs ◽  
Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

scholarly journals Enhancing autophagy by redox regulation extends lifespan in Drosophila

2019 ◽  
Author(s):  
Helena M. Cochemé ◽  
Ivana Bjedov ◽  
Sebastian Grönke ◽  
Katja E. Menger ◽  
Andrew M. James ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Redox Homeostasis ◽  
Model Organism ◽  
Fruit Fly ◽  
Cysteine Residue ◽  
Lifespan Extension ◽  
Post Translational Modification ◽  
Redox Signalling ◽  
Age Related

Redox signalling is an important modulator of diverse biological pathways and processes, and operates through specific post-translational modification of redox-sensitive thiols on cysteine residues 1–4. Critically, redox signalling is distinct from irreversible oxidative damage and functions as a reversible ‘redox switch’ to regulate target proteins. H2O2 acts as the major effector of redox signalling, both directly and through intracellular thiol redox relays 5,6. Dysregulation of redox homeostasis has long been implicated in the pathophysiology of many age-related diseases, as well as in the ageing process itself, however the underlying mechanisms remain largely unclear 7,8. To study redox signalling by H2O2in vivo and explore its involvement in metabolic health and longevity, we used the fruit fly Drosophila as a model organism, with its tractable lifespan and strong evolutionary conservation with mammals 9. Here we report that inducing an endogenous redox-shift, by manipulating levels of the H2O2-degrading enzyme catalase, improves health and robustly extends lifespan in flies, independently of oxidative stress resistance and dietary restriction. We find that the catalase redox-shifted flies are acutely sensitive to starvation stress, which relies on autophagy as a vital survival mechanism. Importantly, we show that autophagy is essential for the lifespan extension of the catalase flies. Furthermore, using redox-inactive knock-in mutants of Atg4a, a major effector of autophagy, we show that the lifespan extension in response to catalase requires a key redox-regulatory cysteine residue, Cys102 in Atg4a. These findings demonstrate that redox regulation of autophagy can extend lifespan, confirming the importance of redox signalling in ageing and as a potential pro-longevity target.

scholarly journals Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

2021 ◽  
Author(s):  
◽  
Jack Alexander Sissons
Keyword(s):  
Single Gene ◽  
Specific Protein ◽  
Rapid Identification ◽  
Phosphopantetheinyl Transferase ◽  
Post Translational Modification ◽  
Diverse Range ◽  
Peptide Motifs ◽  
Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

Abstract 119: Cardiac Transcription Factor EB Sumoylation Deficiency Exacerbates Age-associated Reduction In Autophagy

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Heng Ma ◽  
Le Zhang ◽  
Lu Yu ◽  
Yan Li
Keyword(s):  
Transcription Factor ◽  
Therapeutic Strategy ◽  
Transcriptional Regulator ◽  
Contractile Dysfunction ◽  
Post Translational Modification ◽  
Hairpin Rna ◽  
Adeno Associated Virus ◽  
Transcription Factor Eb

Background: Aging-dependent decline of autophagy contributes to cardiac dysfunction and ischemic intolerance. Transcription factor EB (TFEB) is a master transcriptional regulator of the autophagy-lysosome pathway. The present study aimed to characterize the role of TFEB in the autophagic decrease with aging. Methods and Results: We analyzed age-associated autophagic changes in male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice. The results demonstrated that TFEB expressed predominantly as a SUMOylated form in cardiomyocyte nuclei and this SUMOylation of TFEB declined in aged heart associated with autophagy reduction. Interestingly, SUMOylation of TFEB was unaffected by rapamycin. Rapamycin induced translocation of TFEB into nucleus but lower level of nuclear TFEB in aged hearts than that seen in young hearts (P<0.05). SUMO1 downregulation by adeno-associated-virus-mediated small hairpin RNA (rAAV9-shSUMO1) significantly reduced nuclear TFEB levels (P<0.05), depressed cardiac autophagy and accelerated cardiomyocyte contractile dysfunction with worse hypoxia/reoxygenation (H/R) injury (all P<0.05). Therefore, impaired SUMOylation decreased nuclear TFEB during aging. By contrast, SUMO1 restitution significantly augmented nuclear SUMOylated TFEB with enhanced autophagy and ultimately reduced infarct size in aged heart. However, knockdown of cardiac TFEB blocked the protective effect of upregulation of SUMO1 in aged hearts, resulted in decline of autophagy and worse in vivo I/R injury. Conclusions: The present study newly demonstrates that SUMOylation is a critical post-translational modification that regulates cardiac TFEB. Impaired SUMOylation of TFEB aggravates decline of autophagy in the senescent heart. Targeting SUMO1 may provide a novel therapeutic strategy for the treatment of aging-related loss of cardioprotection.

Comparative Study of Adeno-associated Virus, Adenovirus, Bacu lovirus and Lentivirus Vectors for Gene Therapy of the Eyes

2017 ◽  
Vol 17 (3) ◽  
Author(s):  
Giedrius Kalesnykas ◽  
Emmi Kokki ◽  
Laura Alasaarela ◽  
Hanna P. Lesch ◽  
Timo Tuulos ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Comparative Study ◽  
Adeno Associated Virus ◽  
Lentivirus Vectors

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

scholarly journals In Vivo Expression of Reprogramming Factor OCT4 Ameliorates Myelination Deficits and Induces Striatal Neuroprotection in Huntington’s Disease

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 712
Author(s):  
Ji-Hea Yu ◽  
Bae-Geun Nam ◽  
Min-Gi Kim ◽  
Soonil Pyo ◽  
Jung-Hwa Seo ◽  
...  
Keyword(s):  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Cell Fate ◽  
Gabaergic Neurons ◽  
Oligodendrocyte Progenitor Cells ◽  
Adeno Associated Virus ◽  
In Vivo Expression ◽  
Transcription Factor 4 ◽  
Phosphate Buffered Saline

White matter atrophy has been shown to precede the massive loss of striatal GABAergic neurons in Huntington’s disease (HD). This study investigated the effects of in vivo expression of reprogramming factor octamer-binding transcription factor 4 (OCT4) on neural stem cell (NSC) niche activation in the subventricular zone (SVZ) and induction of cell fate specific to the microenvironment of HD. R6/2 mice randomly received adeno-associated virus 9 (AAV9)-OCT4, AAV9-Null, or phosphate-buffered saline into both lateral ventricles at 4 weeks of age. The AAV9-OCT4 group displayed significantly improved behavioral performance compared to the control groups. Following AAV9-OCT4 treatment, the number of newly generated NSCs and oligodendrocyte progenitor cells (OPCs) significantly increased in the SVZ, and the expression of OPC-related genes and glial cell-derived neurotrophic factor (GDNF) significantly increased. Further, amelioration of myelination deficits in the corpus callosum was observed through electron microscopy and magnetic resonance imaging, and striatal DARPP32+ GABAergic neurons significantly increased in the AAV9-OCT4 group. These results suggest that in situ expression of the reprogramming factor OCT4 in the SVZ induces OPC proliferation, thereby attenuating myelination deficits. Particularly, GDNF released by OPCs seems to induce striatal neuroprotection in HD, which explains the behavioral improvement in R6/2 mice overexpressing OCT4.

scholarly journals Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

2021 ◽  
Author(s):  
Dirk Steinritz ◽  
Robin Lüling ◽  
Markus Siegert ◽  
Julia Herbert ◽  
Harald Mückter ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Dna Adducts ◽  
Sulfur Mustard ◽  
Ex Vivo ◽  
Immunomagnetic Separation ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Islamic State ◽  
Rat Skin

AbstractSulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

scholarly journals N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Ying Dong Du ◽  
Wen Yuan Guo ◽  
Cong Hui Han ◽  
Ying Wang ◽  
Xiao Song Chen ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fragmentation ◽  
Hepatic Ischemia ◽  
Adeno Associated Virus ◽  
Downstream Target ◽  
Hepatic Ischemia Reperfusion

AbstractDespite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.

scholarly journals Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods

2021 ◽  
Vol 22 (13) ◽  
pp. 6696
Author(s):  
Heesu Chae ◽  
Seulki Cho ◽  
Munsik Jeong ◽  
Kiyoung Kwon ◽  
Dongwook Choi ◽  
...  
Keyword(s):  
Computational Methods ◽  
Human Monoclonal Antibody ◽  
Antibody Engineering ◽  
Antigen Binding ◽  
Post Translational Modification ◽  
Biophysical Properties ◽  
Preclinical Development ◽  
Cell Bank ◽  
Aggregation Propensity

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)—which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue—exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.

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