scholarly journals Methods for studying the localization of mitochondrial complexes III and IV by immunofluorescent and immunogold microscopy

2016 ◽  
Vol 68 (4) ◽  
pp. 767-772 ◽  
Author(s):  
Igor Golic ◽  
Marija Aleksic ◽  
Anita Lazarevic ◽  
Maja Bogdanovic ◽  
Slavica Jonic ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Localization ◽  
Mitochondrial Protein ◽  
Microscopic Analysis ◽  
Molecular Architecture ◽  
Protein Protein Interactions ◽  
Chain Complexes ◽  
Bat Mitochondria ◽  
Brown Adipose ◽  
Thin Plastic

The localization of proteins within a cell is very important for studying protein colocalization and subsequently understanding protein-protein interactions at the subcellular level. Using mitochondrial protein localization as a model, we established methods to study the localization of electron transport chain complexes (ETCCs), specifically complexes III and IV, in brown adipose tissue (BAT) and mitochondria. Immunofluorescent and immunogold techniques were applied to BAT paraffin sections and thin Araldite sections of mitochondria-enriched fractions, respectively. Microscopic analysis clearly showed mitochondrial localization of complexes III and IV, as well their colocalization. In addition, 10 and 20 nm gold particles were capable of identifying the localization of complexes within mitochondrial cristae. The methods described in this study may be a beneficial addition to currently utilized methods for accurately identifying the localization of ETCCs, their colocalization with other proteins and their distribution inside the cell and cellular compartments. Lastly, this method can also be used to study the molecular architecture of BAT mitochondria by analyzing fixed and postfixed thin plastic sections with electron microscopy (EM).

scholarly journals Frequent assembly of chimeric complexes in the protein interaction network of an interspecies yeast hybrid

2020 ◽  
Author(s):  
Rohan Dandage ◽  
Caroline M Berger ◽  
Isabelle Gagnon-Arsenault ◽  
Kyung-Mee Moon ◽  
Richard Greg Stacey ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Level ◽  
Yeast Species ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Chimeric Protein ◽  
Interaction Network ◽  
Protein Protein Interactions ◽  
Extreme Phenotypes ◽  
Yeast Hybrid

Abstract Hybrids between species often show extreme phenotypes, including some that take place at the molecular level. In this study, we investigated the phenotypes of an interspecies diploid hybrid in terms of protein-protein interactions inferred from protein correlation profiling. We used two yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum, which are interfertile, but yet have proteins diverged enough to be differentiated using mass spectrometry. Most of the protein-protein interactions are similar between hybrid and parents, and are consistent with the assembly of chimeric complexes, which we validated using an orthogonal approach for the prefoldin complex. We also identified instances of altered protein-protein interactions in the hybrid, for instance in complexes related to proteostasis and in mitochondrial protein complexes. Overall, this study uncovers the likely frequent occurrence of chimeric protein complexes with few exceptions, which may result from incompatibilities or imbalances between the parental proteins.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

scholarly journals The Role of Posttranslational Modifications in DNA Repair

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Miaomiao Bai ◽  
Dongdong Ti ◽  
Qian Mei ◽  
Jiejie Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Protein Localization ◽  
Complex Structure ◽  
Protein Protein Interactions ◽  
Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

Adrenalectomy increases brown adipose tissue metabolism in ob/ob mice housed at 35 degrees C.

1990 ◽  
Vol 259 (3) ◽  
pp. E362
Author(s):  
H K Kim ◽  
D R Romsos
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Mitochondrial Protein ◽  
Mitochondrial Mass ◽  
Warm Environment ◽  
Bat Mitochondria ◽  
Norepinephrine Turnover ◽  
Brown Adipose ◽  
Energy Retention ◽  
High Starch

Adrenalectomy arrests the development of obesity in ob/ob mice fed a high-starch diet and housed at a normal room temperature (20-25 degrees C) partly by stimulating the low thermogenic activity of brown adipose tissue (BAT). The present study was undertaken to determine if adrenalectomy would also lower energy retention and stimulate BAT metabolism in ob/ob mice housed in a warm environment (35 degrees C) where BAT thermoregulatory heat production is not needed. Adrenalectomy prevented hyperphagia and hyperinsulinemia and lowered the efficiency of energy retention in ob/ob mice housed at 35 degrees C, which is comparable to results obtained at 20-25 degrees C. Sympathetic nervous system stimulation of BAT (interscapular and subscapular depots) assessed by norepinephrine turnover was increased in adrenalectomized ob/ob mice. Thermogenic activity of BAT in adrenalectomized ob/ob mice (as assessed by GDP binding to isolated BAT mitochondria, GDP-inhibitable acetate-induced BAT mitochondrial swelling, and Mg2(/)-activated GDP binding to BAT mitochondria) was not elevated when results were expressed per milligram of mitochondrial protein but was elevated approximately 65% when expressed per interscapular and subscapular depots because adrenalectomy increased BAT mitochondrial mass. Adrenalectomy lowers the efficiency of energy retention and stimulates BAT metabolism even when ob/ob mice are housed in a warm environment.

Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes

2000 ◽  
Vol 279 (3) ◽  
pp. C670-C681 ◽  
Author(s):  
J. Himms-Hagen ◽  
A. Melnyk ◽  
M. C. Zingaretti ◽  
E. Ceresi ◽  
G. Barbatelli ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Mitochondrial Protein ◽  
Protein Composition ◽  
Brown Adipocyte ◽  
Brown Adipocytes ◽  
White Adipocytes ◽  
Bat Mitochondria ◽  
Adrenoceptor Agonist ◽  
Brown Adipose ◽  
A Cell

Multilocular, mitochondria-rich adipocytes appear in white adipose tissue (WAT) of rats treated with the β3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes (∼8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.

scholarly journals Cell cycle regulation of mitochondrial protein import revealed by genome-scale pooled bimolecular fluorescence complementation screening

2019 ◽  
Author(s):  
Kim Blakely ◽  
Patricia Mero ◽  
Roland Arnold ◽  
Ayesha Saleem ◽  
Christine Misquitta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Throughput ◽  
Protein Interactions ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Protein Protein Interactions ◽  
Mitochondrial Protein Import ◽  
Interaction Partners ◽  
Genome Scale ◽  
Bimolecular Fluorescence

ABSTRACTA central focus of systems biology is the functional mapping of protein-protein interactions under physiological conditions. Here we describe MaGiCaL-BiFC, a lentivirus-based bimolecular fluorescence protein-fragment complementation approach for the high-throughput, genome-scale identification of protein-protein interactions in mammalian cells. After developing and validating this methodology using known protein-protein interaction pairs, we constructed genome-scale pooled BiFC libraries using the human ORFeome cDNA collection. These pooled libraries, containing ∼ 12,000 unique human cDNAs, were used to screen for candidate interaction partners of the mitochondrial transmembrane protein TOMM22. Following infection of cells with the TOMM22 bait and the pooled cDNA libraries, cells harboring candidate TOMM22 interacting proteins were isolated from the cell pool via fluorescence activated cell sorting, and identified via microarray analysis. This approach identified several known interaction partners of TOMM22, as well as novel physical and functional partners that link the mitochondrial network to proteins involved in diverse cellular processes. Notably, protein kinase CK2 was identified as a novel physical interaction partner of human TOMM22. We found that this association occurs preferentially during mitosis and involves direct phosphorylation of TOMM22, an event that may lead to attenuation of mitochondrial protein import. Together, this data contributes to the growing body of evidence suggesting eloquent coordination between cell cycle progression and mitochondrial physiology. Importantly, through high-throughput screening and focused validation, our study demonstrates the power of the MaGiCaL-BiFC approach to uncover novel functional protein-protein interactions, including those involving proteins with membrane-spanning domains, or of a transient nature, all within their native cellular environment.

scholarly journals Human mitochondrial protein complexes revealed by large-scale coevolution analysis and deep learning-based structure modeling

2021 ◽  
Author(s):  
Jimin Pei ◽  
Jing Zhang ◽  
Qian Cong
Keyword(s):  
Deep Learning ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Protein Structures ◽  
Complex Structures ◽  
Protein Protein Interactions ◽  
Learning Methods ◽  
Contact Probability

AbstractRecent development of deep-learning methods has led to a breakthrough in the prediction accuracy of 3-dimensional protein structures. Extending these methods to protein pairs is expected to allow large-scale detection of protein-protein interactions and modeling protein complexes at the proteome level. We applied RoseTTAFold and AlphaFold2, two of the latest deep-learning methods for structure predictions, to analyze coevolution of human proteins residing in mitochondria, an organelle of vital importance in many cellular processes including energy production, metabolism, cell death, and antiviral response. Variations in mitochondrial proteins have been linked to a plethora of human diseases and genetic conditions. RoseTTAFold, with high computational speed, was used to predict the coevolution of about 95% of mitochondrial protein pairs. Top-ranked pairs were further subject to the modeling of the complex structures by AlphaFold2, which also produced contact probability with high precision and in many cases consistent with RoseTTAFold. Most of the top ranked pairs with high contact probability were supported by known protein-protein interactions and/or similarities to experimental structural complexes. For high-scoring pairs without experimental complex structures, our coevolution analyses and structural models shed light on the details of their interfaces, including CHCHD4-AIFM1, MTERF3-TRUB2, FMC1-ATPAF2, ECSIT-NDUFAF1 and COQ7-COQ9, among others. We also identified novel PPIs (PYURF-NDUFAF5, LYRM1-MTRF1L and COA8-COX10) for several proteins without experimentally characterized interaction partners, leading to predictions of their molecular functions and the biological processes they are involved in.

scholarly journals Tau Post-translational Modifications: Dynamic Transformers of Tau Function, Degradation, and Aggregation

2021 ◽  
Vol 11 ◽  
Author(s):  
Carolina Alquezar ◽  
Shruti Arya ◽  
Aimee W. Kao
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Protein Localization ◽  
Critical Role ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Tau Function ◽  
Potential Impact ◽  
Important Position

Post-translational modifications (PTMs) on tau have long been recognized as affecting protein function and contributing to neurodegeneration. The explosion of information on potential and observed PTMs on tau provides an opportunity to better understand these modifications in the context of tau homeostasis, which becomes perturbed with aging and disease. Prevailing views regard tau as a protein that undergoes abnormal phosphorylation prior to its accumulation into the toxic aggregates implicated in Alzheimer's disease (AD) and other tauopathies. However, the phosphorylation of tau may, in fact, represent part of the normal but interrupted function and catabolism of the protein. In addition to phosphorylation, tau undergoes another forms of post-translational modification including (but not limited to), acetylation, ubiquitination, glycation, glycosylation, SUMOylation, methylation, oxidation, and nitration. A holistic appreciation of how these PTMs regulate tau during health and are potentially hijacked in disease remains elusive. Recent studies have reinforced the idea that PTMs play a critical role in tau localization, protein-protein interactions, maintenance of levels, and modifying aggregate structure. These studies also provide tantalizing clues into the possibility that neurons actively choose how tau is post-translationally modified, in potentially competitive and combinatorial ways, to achieve broad, cellular programs commensurate with the distinctive environmental conditions found during development, aging, stress, and disease. Here, we review tau PTMs and describe what is currently known about their functional impacts. In addition, we classify these PTMs from the perspectives of protein localization, electrostatics, and stability, which all contribute to normal tau function and homeostasis. Finally, we assess the potential impact of tau PTMs on tau solubility and aggregation. Tau occupies an undoubtedly important position in the biology of neurodegenerative diseases. This review aims to provide an integrated perspective of how post-translational modifications actively, purposefully, and dynamically remodel tau function, clearance, and aggregation. In doing so, we hope to enable a more comprehensive understanding of tau PTMs that will positively impact future studies.

Frataxin, a molecule of mystery: trading stability for function in its iron-binding site

2010 ◽  
Vol 426 (2) ◽  
pp. e1-e3 ◽  
Author(s):  
Darius J. R. Lane ◽  
Des R. Richardson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Mitochondrial Protein ◽  
Iron Binding ◽  
Protein Protein Interactions ◽  
Iron Sulfur Cluster ◽  
Downstream Effects ◽  
Biochemical Journal ◽  
And Function ◽  
The Relationship

What are the structural implications for iron binding by frataxin, the mitochondrial protein whose decreased expression results in Friedreich's ataxia? Though frataxin has been shown to be essential for proper handling of iron within mitochondria (e.g. for iron–sulfur cluster and haem biosynthesis), its exact molecular function remains unclear. In this issue of the Biochemical Journal, Correia and colleagues investigate the relationship between structure and function at the putative iron-binding site of Yfh1 (yeast frataxin). Using a host of Yfh1 combination point mutants, the authors observe that the presence of a semi-conserved pocket of negative charge within the ‘acidic ridge’ region (thought to be responsible for iron binding) only mildly enhances Yfh1's ability to bind iron, though it does significantly increase the protein's structural flexibility. The general emerging view is that frataxin's keystone role in mitochondrial iron metabolism depends on iron binding. This appears to have downstream effects on protein–protein interactions that are crucial for frataxin function. The current results reveal a somewhat delicate relationship between iron binding and structural plasticity that may help unravel the enigma of frataxin's metabolic roles.

scholarly journals Synthetic protein interactions reveal a functional map of the cell

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lisa K Berry ◽  
Guðjón Ólafsson ◽  
Elena Ledesma-Fernández ◽  
Peter H Thorpe
Keyword(s):  
Protein Interactions ◽  
Protein Localization ◽  
Eukaryotic Cells ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Synthetic Protein ◽  
Protein Functions ◽  
Functional Map ◽  
Cellular Compartments ◽  
New Protein

To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells.

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