scholarly journals Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1741
Author(s):  
Carla M. Kumbale ◽  
Eberhard O. Voit ◽  
Qiang Zhang
Keyword(s):  
Signal Amplification ◽  
Biological Rhythms ◽  
Covalent Modification ◽  
Protein Stabilization ◽  
Enzymatic Reactions ◽  
Signaling Proteins ◽  
Protein Substrate ◽  
Free Protein ◽  
Protein Substrates ◽  
Inducible Protein

Signal amplification in biomolecular networks converts a linear input to a steeply sigmoid output and is central to a number of cellular functions including proliferation, differentiation, homeostasis, adaptation, and biological rhythms. One canonical signal amplifying motif is zero-order ultrasensitivity that is mediated through the posttranslational modification (PTM) cycle of signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant, as by the classical Koshland–Goldbeter model. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here, we use mathematical models to explore the signal amplification properties in such scenarios and report some novel aspects. Our analyses indicate that PTM-induced protein stabilization brings the enzymes closer to saturation. As a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response, higher magnitude, and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Importantly, ultrasensitivity is not limited to modified or unmodified protein substrates—when protein turnover is considered, the total free protein substrate can also exhibit ultrasensitivity under several conditions. When full enzymatic reactions are used instead of Michaelis–Menten kinetics for the modeling, the total free protein substrate can even exhibit nonmonotonic dose–response patterns. It is conceivable that cells use inducible protein stabilization as a strategy in the signaling network to boost signal amplification while saving energy by keeping the protein substrate levels low at basal conditions.

scholarly journals Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

2021 ◽  
Author(s):  
Carla Kumbale ◽  
Eberhard Voit ◽  
Qiang Zhang
Keyword(s):  
Posttranslational Modification ◽  
Signal Amplification ◽  
Covalent Modification ◽  
Zero Order ◽  
Protein Stabilization ◽  
Signaling Proteins ◽  
Cellular Functions ◽  
Protein Substrate ◽  
Protein Substrates ◽  
Inducible Protein

Signal amplification converts a linear input to a steeply sigmoid output and is central to cellular functions. One canonical signal amplifying motif is zero-order ultrasensitivity through the posttranslational modification (PTM) cycle signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here we use a mathematical model to explore the signal amplification properties in such scenarios. Our simulations indicate that PTM-induced protein stabilization brings the enzymes closer to saturation, and as a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response of higher magnitude and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Interestingly, ultrasensitivity is not limited to modified or unmodified protein substrates; the total protein substrate can also exhibit ultrasensitivity. It is conceivable that cells use inducible protein stabilization as a way to boost signal amplification while saving energy by keeping the protein substrate at low basal conditions.

scholarly journals Mieap forms membraneless organelles to compartmentalize and facilitate cardiolipin metabolism

2020 ◽  
Author(s):  
Naoki Ikari ◽  
Katsuko Honjo ◽  
Yoko Sagami ◽  
Yasuyuki Nakamura ◽  
Hirofumi Arakawa
Keyword(s):  
Quality Control ◽  
Critical Role ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Quality Control ◽  
Kidney Mitochondria ◽  
Intrinsically Disordered ◽  
Liver And Kidney ◽  
Inducible Protein

AbstractLiquid droplets function as membraneless organelles that compartmentalize and facilitate efficient biological reactions in cells. They are formed by proteins with an intrinsically disordered region(s) (IDR) via liquid–liquid phase separation. Mieap/SPATA18, a p53-inducible protein, plays a critical role in the suppression of human and murine colorectal tumors via mitochondrial quality control. However, the regulatory mechanism underlying this process remains unclear. Here, we report that Mieap is an IDR-containing protein that drives the formation of liquid droplets in the mitochondria. Mieap liquid droplets (MLDs) specifically phase separate the mitochondrial phospholipid cardiolipin. Lipidomic analysis of cardiolipin suggested that Mieap promotes enzymatic reactions involved in cardiolipin metabolism, including biosynthesis and remodeling. Accordingly, four cardiolipin biosynthesis enzymes, TAMM41, PGS1, PTPMT1, and CRLS1, and two remodeling enzymes, PLA2G6 and TAZ, are phase separated by MLDs. Mieap-deficient mice exhibited altered cristae structure in the liver and kidney mitochondria and a trend of obesity. These results suggest that Mieap drives the formation of membraneless organelles to compartmentalize and promotes cardiolipin metabolism at the inner mitochondrial membrane, thus playing a possible role in mitochondrial quality control.

scholarly journals McSNAC: A software to approximate first-order signaling networks from mass cytometry data

2021 ◽  
Author(s):  
Darren Wethington ◽  
Sayak Mukherjee ◽  
Jayajit Das
Keyword(s):  
Single Cell ◽  
In Silico ◽  
Single Cells ◽  
Signaling Network ◽  
Signaling Networks ◽  
Enzymatic Reactions ◽  
Signaling Proteins ◽  
Protein Species ◽  
Mass Cytometry ◽  
First Order

AbstractMass cytometry (CyTOF) gives unprecedented opportunity to simultaneously measure up to 40 proteins in single cells, with a theoretical potential to reach 100 proteins. This high-dimensional single-cell information can be very useful to dissecting mechanisms of cellular activity. In particular, measuring abundances of signaling proteins like phospho-proteins can provide detailed information on the dynamics of single-cell signaling processes. With a proper computational analysis, timestamped CyTOF data of signaling proteins could help develop predictive and mechanistic models for signaling kinetics. These models would be useful for predicting the effects of perturbations in cells, or comparing signaling networks across cell groups. We propose our Mass cytometry Signaling Network Analysis Code, or McSNAC, a new software capable of reconstructing signaling networks and estimating their kinetic parameters from CyTOF data.McSNAC approximates signaling networks as a network of first-order reactions between proteins. This assumption breaks down often as signaling reactions can involve binding and unbinding, enzymatic reactions, and other nonlinear constructions. Furthermore, McSNAC may be limited to approximating indirect interactions between protein species, as cytometry experiments are only able to assay a small fraction of the protein species that are involved in signaling. We carry out a series of in silico experiments here to show that 1) McSNAC is capable of accurately estimating the ground-truth model in a scalable manner when given data originating from a first-order system; 2) McSNAC is capable of qualitatively predicting outcomes to perturbations of species abundances in simple second-order reaction models and in a complex in silico nonlinear signaling network in which some proteins are unmeasured. These findings demonstrate that McSNAC can be a valuable screening tool for generating models of signaling networks from timestamped CyTOF data.

scholarly journals Characteristics of polyamine stimulation of cyclic nucleotide-independent protein kinase reactions

1985 ◽  
Vol 232 (3) ◽  
pp. 767-771 ◽  
Author(s):  
K Ahmed ◽  
S A Goueli ◽  
H G Williams-Ashman
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Nuclear Protein ◽  
Direct Stimulation ◽  
Protein Substrate ◽  
Protein Substrates ◽  
Effects Of Temperature ◽  
Independent Protein ◽  
Stimulation Of

The extent of direct stimulation by spermine of reactions catalysed by nuclear N1 and N2 protein kinases purified from liver and prostate depends critically on the nature of the protein substrate. The chemically inert Co(NH3)36+ ion exerts effects on protein kinase reactions similar to those of spermidine or spermine. This enhancement of the phosphorylation of various protein substrates by polyamines or Co(NH3)63+ by purified nuclear protein kinase preparations was studied in relation to effects of temperature, pH and other factors. The results provide further support for our hypothesis [Ahmed, Wilson, Goueli & Williams-Ashman (1978) Biochem. J. 176, 739-750] that the enhancement of certain protein kinase reactions by polycations relates primarily to their interaction with the protein substrate, yielding more favourable conformations for phosphorylation by the protein kinase, rather than a direct effect on its catalytic activity.

scholarly journals Highly multiplexedin situprotein imaging with signal amplification by Immuno-SABER

2018 ◽  
Author(s):  
Sinem K. Saka ◽  
Yu Wang ◽  
Jocelyn Y. Kishi ◽  
Allen Zhu ◽  
Yitian Zeng ◽  
...  
Keyword(s):  
Exchange Reaction ◽  
Signal Amplification ◽  
Cultured Cells ◽  
Molecular Organization ◽  
Tissue Imaging ◽  
Enzymatic Reactions ◽  
Multiple Targets ◽  
Standard Equipment ◽  
Major Barrier

AbstractProbing the molecular organization of tissues requiresin situanalysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets withoutin situenzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

scholarly journals Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification

2018 ◽  
Vol 116 (3) ◽  
pp. 810-815 ◽  
Author(s):  
Nicole A. Perry ◽  
Tamer S. Kaoud ◽  
Oscar O. Ortega ◽  
Ali I. Kaya ◽  
David J. Marcus ◽  
...  
Keyword(s):  
Bayesian Inference ◽  
Signal Amplification ◽  
Conveyor Belt ◽  
Computational Approach ◽  
Scaffold Proteins ◽  
Signaling Cascade ◽  
Signaling Proteins ◽  
Catalytic Phosphorylation ◽  
Upstream Kinase ◽  
Phosphorylation Rate

Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a “conveyor belt” mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

1988 ◽  
Vol 173 (1) ◽  
pp. 147-154 ◽  
Author(s):  
Vadim V. MOZHAEV ◽  
Virginius A. SIKSNIS ◽  
Nikolay S. MELIK-NUBAROV ◽  
Nida Z. GALKANTAITE ◽  
Gervydas J. DENIS ◽  
...  
Keyword(s):  
Covalent Modification ◽  
Protein Stabilization

Catabolism of intracellular protein: molecular aspects

1986 ◽  
Vol 251 (2) ◽  
pp. C141-C152 ◽  
Author(s):  
R. J. Beynon ◽  
J. S. Bond
Keyword(s):  
Mammalian Cells ◽  
Specific Protein ◽  
Covalent Modification ◽  
Protein Substrates ◽  
Initial Event ◽  
Great Heterogeneity ◽  
Ubiquitin Conjugation ◽  
Extensive Degradation ◽  
Molecular Aspects

All living cells regulate the content and composition of their resident proteins, but the mechanisms by which this is accomplished are not understood. The process of protein degradation has an important role in determining steady state and fluctuations of protein concentrations in mammalian cells. This process may be regulated by innate properties of the protein substrates, by factors that interact or "brand" proteins for degradation or by the degradative machinery of the cell. For a specific protein, there appears to be a committed step, an irreversible event that leads to rapid and extensive degradation. That initial event may or may not involve 1) proteolysis, 2) a nonproteolytic covalent modification or branding event (e.g., oxidation, ubiquitin conjugation), 3) denaturation or unfolding of the protein, or 4) sequestration. The degradative machinery of cells may either recognize proteins committed to degradation or initiate degradation, but the process must be selective because there is great heterogeneity in the rates of degradation for different proteins of one cell. The degradative process certainly requires proteases, and it is probable that lysosomal and extralysosomal proteases are involved in the catabolism of cellular proteins. We review here briefly what is currently known about the factors that may determine the half-life of a protein in a mammalian cell, the role of the protein substrate and sequestration in the process, the proteolytic and nonproteolytic enzymes that may initiate the degradative process, and the regulation of extensive degradation of proteins in cells.

scholarly journals Mieap forms membraneless organelles to compartmentalize and facilitate cardiolipin metabolism

2021 ◽  
Author(s):  
Naoki Ikari ◽  
Katsuko Honjo ◽  
Yoko Sagami ◽  
Yasuyuki Nakamura ◽  
Hirofumi Arakawa
Keyword(s):  
Quality Control ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Quality Control ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Inducible Protein ◽  
Deficient Mice ◽  
Liquid Liquid Phase Separation

Abstract Liquid droplets function as membraneless organelles that compartmentalize and facilitate efficient biological reactions. They are formed by proteins with intrinsically disordered regions (IDRs) via liquid–liquid phase separation. Mieap/SPATA18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of liquid droplets in mitochondria. Mieap liquid droplets specifically phase separate the mitochondrial phospholipid, cardiolipin. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions involved in cardiolipin metabolism, including biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1, and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap liquid droplets. Mieap-deficient mice exhibit altered crista structure in mitochondria of various tissues, including brown fat, and tend to become obese. These results suggest that Mieap drives formation of membraneless organelles to compartmentalize and promote cardiolipin metabolism at the inner mitochondrial membrane, thus potentially contributing to mitochondrial quality control.

scholarly journals The Many Potential Fates of Non-Canonical Protein Substrates Subject to NEDDylation

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2660
Author(s):  
Kartikeya Vijayasimha ◽  
Brian P. Dolan
Keyword(s):  
Cancer Progression ◽  
Ring Finger ◽  
Eukaryotic Cell ◽  
Covalent Modification ◽  
Protein Substrates ◽  
Rectal Cancers ◽  
The Many ◽  
Neuronal Precursor Cell

Neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) is a ubiquitin-like protein (UBL) whose canonical function involves binding to, and thus, activating Cullin–Ring finger Ligases (CRLs), one of the largest family of ubiquitin ligases in the eukaryotic cell. However, in recent years, several non-canonical protein substrates of NEDD8 have been identified. Here we attempt to review the recent literature regarding non-canonical NEDDylation of substrates with a particular focus on how the covalent modification of NEDD8 alters the protein substrate. Like much in the study of ubiquitin and UBLs, there are no clear and all-encompassing explanations to satisfy the textbooks. In some instances, NEDD8 modification appears to alter the substrates localization, particularly during times of stress. NEDDylation may also have conflicting impacts upon a protein’s stability: some reports indicate NEDDylation may protect against degradation whereas others show NEDDylation can promote degradation. We also examine how many of the in vitro studies measuring non-canonical NEDDylation were conducted and compare those conditions to those which may occur in vivo, such as cancer progression. It is likely that the conditions used to study non-canonical NEDDylation are similar to some types of cancers, such as glioblastoma, colon and rectal cancers, and lung adenocarcinomas. Although the full outcomes of non-canonical NEDDylation remain unknown, our review of the literature suggests that researchers keep an open mind to the situations where this modification occurs and determine the functional impacts of NEDD8-modification to the specific substrates which they study.

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