scholarly journals Exploring absent protein function in yeast: assaying post translational modification and human genetic variation

2021 ◽  
Vol 8 (8) ◽  
pp. 164-183
Author(s):  
Christina S. Moesslacher ◽  
Johanna M. Kohlmayr ◽  
Ulrich Stelzl
Keyword(s):  
Protein Function ◽  
Model Organism ◽  
Global Scale ◽  
Test System ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Protein Modifications ◽  
In Vivo Test ◽  
The Impact

Yeast is a valuable eukaryotic model organism that has evolved many processes conserved up to humans, yet many protein functions, including certain DNA and protein modifications, are absent. It is this absence of protein function that is fundamental to approaches using yeast as an in vivo test system to investigate human proteins. Functionality of the heterologous expressed proteins is connected to a quantitative, selectable phenotype, enabling the systematic analyses of mechanisms and specificity of DNA modification, post-translational protein modifications as well as the impact of annotated cancer mutations and coding variation on protein activity and interaction. Through continuous improvements of yeast screening systems, this is increasingly carried out on a global scale using deep mutational scanning approaches. Here we discuss the applicability of yeast systems to investigate absent human protein function with a specific focus on the impact of protein variation on protein-protein interaction modulation.

scholarly journals Photocontrollable PROTAC molecules: Structure and mechanism of action

2021 ◽  
Vol 71 (3) ◽  
pp. 161-176
Author(s):  
Mladen Koravović ◽  
Gordana Tasić ◽  
Milena Rmandić ◽  
Bojan Marković
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Binding Sites ◽  
E3 Ubiquitin Ligase ◽  
Drug Binding ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Target Drug ◽  
Traditional Drug

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

scholarly journals Development of a Mouse Glioblastoma Orthotopic Model Using the GLi-261 Cell Line

2019 ◽  
Vol 19 (4) ◽  
pp. 242-250
Author(s):  
A. A. Borzov ◽  
A. A. Оvsepyan ◽  
E. I. Katorkina ◽  
E. O. Anisimova ◽  
M. V. Lykov
Keyword(s):  
Brain Tumor ◽  
Magnetic Resonance ◽  
Neurological Status ◽  
Tumour Volume ◽  
Test System ◽  
Experimental Models ◽  
Orthotopic Model ◽  
In Vivo Test

Glioblastoma is the most common and most aggressive type of brain tumor, with an almost 100 % mortality rate over 5 years. The search for new effective approaches to the treatment of this disease requires the development of adequate experimental models.Objective: to develop and put into practice an orthotopic model of mouse glioblastoma.Materials and methods: GLi-261 mouse glioma cells were orthotopically inoculated into the putamen of C57Bl/6 mice brain. Tumor dynamics was investigated by Preclinical MRI System 7.0T/17cm (Flexiscan) highfield magnetic resonance imager (MR Solutions, UK). Temcital® (temozolomide) was used as a positive control in the treatment of experimental glioblastoma. The neurological status of animals in the course of tumour development was assessed by specific tests.Results: a GLi-261 cell-based mouse glioblastoma orthotopic model was developed using stereotactic equipment for accurate inoculation of tumour cells, magnetic resonance imaging for non-invasive determination of tumour volume and dynamics, and special tests for determination of the neurological status of the biological test systems. This model was used to demonstrate the effectiveness of temozolomide (the «gold standard» for glioblastoma treatment).Conclusions: this model has been introduced into practice at the IBC Generium, LLC, and can be used as an in vivo test system for preclinical evaluation of efficacy of new antitumour drugs being developed, as well as brain cancer treatment regimens using combination therapy.

scholarly journals Blockchains for Supply Chain Management: Architectural Elements and Challenges Towards a Global Scale Deployment

Logistics ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 5 ◽  
Author(s):  
Antonios Litke ◽  
Dimosthenis Anagnostopoulos ◽  
Theodora Varvarigou
Keyword(s):  
Supply Chain ◽  
Supply Chain Management ◽  
Global Scale ◽  
Chain Management ◽  
Architectural Elements ◽  
Blockchain Technology ◽  
High Level ◽  
The Impact ◽  
Digital Assets

Blockchains are attracting the attention of stakeholders in many industrial domains, including the logistics and supply chain industries. Blockchain technology can effectively contribute in recording every single asset throughout its flow on the supply chain, contribute in tracking orders, receipts, and payments, while track digital assets such as warranties and licenses in a unified and transparent way. The paper provides, through its methodology, a detailed analysis of the blockchain fit in the supply chain industry. It defines the specific elements of blockchain that affect supply chain such as scalability, performance, consensus mechanism, privacy considerations, location proof and cost, and details on the impact that blockchains will have in disrupting the supply chain industry. Discussing the tradeoff between consensus cost, throughput and validation time it proceeds with a suggested high-level architectural approach, and concludes as a result with a discussion on changes needed and challenges faced for an in-vivo deployment of blockchains in the supply chain industry. While the technological features of modern blockchains can effectively facilitate supply chain uses cases, the various challenges that still remain, bring in front of us a wide set of needed changes and further research efforts for achieving a global, production level blockchain for the supply chain industry.

scholarly journals SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

Open Biology ◽  
2013 ◽  
Vol 3 (10) ◽  
pp. 120173 ◽  
Author(s):  
Ingrid Kassner ◽  
Anneli Andersson ◽  
Monika Fey ◽  
Martin Tomas ◽  
Elisa Ferrando-May ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Interactions ◽  
Protein Function ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Protein Methyltransferase

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo . ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo . Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

scholarly journals fuNTRp: Identifying protein positions for variation driven functional tuning

2019 ◽  
Author(s):  
Maximilian Miller ◽  
Daniel Vitale ◽  
Peter Kahn ◽  
Burkhard Rost ◽  
Yana Bromberg
Keyword(s):  
Protein Design ◽  
Computational Method ◽  
Protein Activity ◽  
Protein Functions ◽  
Disease Associations ◽  
Position Type ◽  
Variant Effect ◽  
Evolution Understanding ◽  
The Impact

ABSTRACTEvaluating the impact of non-synonymous genetic variants is essential for uncovering disease associations and mechanisms of evolution. Understanding corresponding sequence changes is also fundamental for synthetic protein design and stability assessments. However, the performance gain of variant effect predictors observed in recent years is not in line with the increased complexity of new methods. One likely reason for this might be that most approaches use similar sets of gene/protein features for modeling variant effect, often emphasizing sequence conservation. While high levels of conservation highlight residues essential for protein activity, much of the in vivo observable variation is arguably weaker in its impact and, thus, requires evaluation at a higher level of resolution. Here we describe function Neutral/Toggle/Rheostat predictor (funtrp), a novel computational method that categorizes protein positions based on the position-specific expected range of mutational impacts: Neutral (weak/no effects), Rheostat (function-tuning positions), or Toggle (on/off switches). We show that position types do not correlate strongly with familiar protein features such as conservation or protein disorder. We also find that position type distribution varies across different protein functions. Finally, we demonstrate that position types reflect experimentally determined functional effects and can thus improve performance of existing variant effect predictors and suggest a way forward for the development of new ones.

scholarly journals Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jugal Mohapatra ◽  
Kyuto Tashiro ◽  
Ryan L Beckner ◽  
Jorge Sierra ◽  
Jessica A Kilgore ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Protein Function ◽  
Post Translational Modification ◽  
Site Specific ◽  
Precise Control ◽  
Protein Ligation ◽  
Adp Ribosylation ◽  
Nuclear Extracts ◽  
Synthesis Strategy ◽  
The Impact

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

USE OF AN IN VIVO TEST SYSTEM TO INVESTIGATE THE ACUTE AND SUB-ACUTE RESPONSES OF THE RAT LUNG TO MINERAL DUSTS

1982 ◽  
pp. 593-605
Author(s):  
S.E. SYKES ◽  
A. MORGAN ◽  
J.C. EVANS ◽  
N. EVANS ◽  
A. HOLMES ◽  
...  
Keyword(s):  
Test System ◽  
Rat Lung ◽  
In Vivo Test ◽  
Acute Responses ◽  
Mineral Dusts ◽  
In Vivo Test System

Influence of silver-containing filler on antibacterial properties of experimental resin composites against Enterococcus faecalis

2021 ◽  
Vol 109 (2) ◽  
pp. 59-67
Author(s):  
Sanadra Adeeb ◽  
Saja Adeeb ◽  
G. Chladek ◽  
W. Pakieła ◽  
A. Mertas
Keyword(s):  
Enterococcus Faecalis ◽  
Test Performance ◽  
Antibacterial Properties ◽  
Antimicrobial Properties ◽  
Positive Control ◽  
Control Material ◽  
In Vivo Test ◽  
Laboratory Test Results ◽  
The Impact

Purpose: The aim of the presented work was to investigate the impact of the S-P introduction into resin-based composites on their effectiveness against Enterococcus faecalis (E. faecalis). Design/methodology/approach: Seven experimental composites based on typical matrix were developed. Six of them contained a filler with antimicrobial properties (silver sodium hydrogen zirconium phosphate, S-P), while the control material contained only common reinforcement fillers. The materials were characterized in terms of the dispersion of the extender in the matrix and then subjected to microbiological tests. The efficiency in the reduction of E. faecalis in the microenvironment was tested. Findings: The composites show a satisfactory distribution of fillers and a high initial reduction of bacteria colonies for the tested strain of E. faecalis. The reduction in bacteria colonies achieved for S-P concentrations from 7% to 13% was similar (median value from 99.8 to 99.9%, when for control material and compound with 1% S-P the number of colonies increased compared to positive control. Research limitations/implications: Laboratory test results may differ from in vivo test performance. In addition, there are many models for conducting laboratory antimicrobial efficacy studies, the results of which are also varied. The cytotoxic tests, long-term investigations and in vivo experiments need to be performed in future experiments. Practical implications: E. faecalis is a Gram-positive bacterium that is commonly detected in persistent endodontic infections and may enter the root canal through the coronal part. Development of composites with antimicrobial properties against this bacterium is as important as obtaining efficacy against cariogenic bacteria. Originality/value: The antimicrobial effectiveness against E. faecalis of experimental composites with submicrometer-sized particles of S-P was not investigated until now.

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