protein substrates
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2022 ◽  
Author(s):  
Tatyana Pivnenko ◽  
Julia Karpenko ◽  
Viktoria Krashchenko

This research aimed to examine the effect of a microbial transglutaminase preparation (Activa® GS, Ajinomoto Co., Inc, Japan) on the structure formation of the myofibrillar protein system of a deep-sea fish species – the giant grenadier – after the addition of various protein substrates. The low content of proteins and their low water-holding capacity in this fish, subjected to various processing methods, leads to significant losses in the initial mass and decreased gelation ability in the muscle tissue system. Various concentrations of transglutaminase were used but these did not ensure the restructuring of the initial muscle tissue of the grenadier. Additional protein substrates with different molecular weights and amino acid composition were added, including gelatin, milk casein, hydrolysates of the skin and milt of the fish, and whole bivalves, which were used to create a firm structure. It was shown that the introduction of gelatin and casein at a concentration of 5% led to the formation of a firm, thermostable structure under the action of the enzyme, while hydrolyzed proteins with low molecular weight at their various concentrations enhanced the expression of water and formation of the fluid consistency. The ability of gastrointestinal tract proteases (pepsin and trypsin) to digest did not depend on the formation of protein-to-protein cross-linking in these combined products. The influence on the growth of the Tetrahymena pyriformis ciliates test culture also showed the high degree of product availability. The technology of molded products based on fermented minced muscle tissue of grenadier with added casein, both in the form of semi-finished products and in the form of ready-to-eat products, was developed. Keywords: transglutaminase, muscle tissue, structure formation, deep-sea fish


Author(s):  
Ana Kasirer-Friede ◽  
Emilia Peuhu ◽  
Johanna Ivaska ◽  
Sanford J. Shattil

Platelets form hemostatic plugs to prevent blood loss and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule, SHARPIN, that can associate directly with the αIIb cytoplasmic tail and can separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < 0.01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to sub-maximal concentrations of ADP (P < 0.05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classical platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < 0.01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.


2021 ◽  
Vol 23 (1) ◽  
pp. 84
Author(s):  
Rubi Campos Gudiño ◽  
Ally C. Farrell ◽  
Nicole M. Neudorf ◽  
Kirk J. McManus

The SKP1, CUL1, F-box protein (SCF) complex represents a family of 69 E3 ubiquitin ligases that poly-ubiquitinate protein substrates marking them for proteolytic degradation via the 26S proteasome. Established SCF complex targets include transcription factors, oncoproteins and tumor suppressors that modulate cell cycle activity and mitotic fidelity. Accordingly, genetic and epigenetic alterations involving SCF complex member genes are expected to adversely impact target regulation and contribute to disease etiology. To gain novel insight into cancer pathogenesis, we determined the prevalence of genetic and epigenetic alterations in six prototypic SCF complex member genes (SKP1, CUL1, RBX1, SKP2, FBXW7 and FBXO5) from patient datasets extracted from The Cancer Genome Atlas (TCGA). Collectively, ~45% of observed SCF complex member mutations are predicted to impact complex structure and/or function in 10 solid tumor types. In addition, the distribution of encoded alterations suggest SCF complex members may exhibit either tumor suppressor or oncogenic mutational profiles in a cancer type dependent manner. Further bioinformatic analyses reveal the potential functional implications of encoded alterations arising from missense mutations by examining predicted deleterious mutations with available crystal structures. The SCF complex also exhibits frequent copy number alterations in a variety of cancer types that generally correspond with mRNA expression levels. Finally, we note that SCF complex member genes are differentially methylated across cancer types, which may effectively phenocopy gene copy number alterations. Collectively, these data show that SCF complex member genes are frequently altered at the genetic and epigenetic levels in many cancer types, which will adversely impact the normal targeting and timely destruction of protein substrates, which may contribute to the development and progression of an extensive array of cancer types.


2021 ◽  
Author(s):  
Rufus Hards ◽  
Charles L. Howarth ◽  
Kwame Wiredu ◽  
Ian LaCroix ◽  
Juan Carlos Mercado del Valle ◽  
...  

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.


2021 ◽  
Author(s):  
Zhejian Ji ◽  
Hao Li ◽  
Daniele Peterle ◽  
Joao A. Paulo ◽  
Scott B. Ficarro ◽  
...  
Keyword(s):  

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1741
Author(s):  
Carla M. Kumbale ◽  
Eberhard O. Voit ◽  
Qiang Zhang

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.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Supawadee Maneekesorn ◽  
Ellen Knuepfer ◽  
Judith L. Green ◽  
Parichat Prommana ◽  
Chairat Uthaipibull ◽  
...  

AbstractThe inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth.


Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1090
Author(s):  
Rumi Mikami ◽  
Shunsuke Tsukagoshi ◽  
Kenta Arai

In a previous study, we reported that (S)-1,2-diselenane-4-amine (1) catalyzes oxidative protein folding through protein disulfide isomerase (PDI)-like catalytic mechanisms and that the direct conjugation of a basic amino acid (Xaa: His, Lys, or Arg) via an amide bond improves the catalytic activity of 1 by increasing its diselenide (Se–Se) reduction potential (E′°). In this study, to modulate the Se–Se redox properties and the association of the compounds with a protein substrate, new catalysts, in which a Gly spacer was inserted between 1 and Xaa, were synthesized. Exhaustive comparison of the PDI-like catalytic activities and E′° values among 1, 1-Xaa, and 1-Gly-Xaa showed that the insertion of a Gly spacer into 1-Xaa either did not change or slightly reduced the PDI-like activity and the E′° values. Importantly, however, only 1-Gly-Arg deviated from this generality and showed obviously increased E°′ value and PDI-like activity compared to the corresponding compound with no Gly spacer (1-Arg); on the contrary, its catalytic activity was the highest among the diselenide compounds employed in this study, while this abnormal enhancement of the catalytic activity of 1-Gly-Arg could not be fully explained by the thermodynamics of the Se–Se bond and its association ability with protein substrates.


2021 ◽  
Author(s):  
Zhejian Ji ◽  
Hao Li ◽  
Daniele Peterle ◽  
Joao A Paulo ◽  
Scott B Ficarro ◽  
...  

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.


2021 ◽  
Author(s):  
Alan Cowman ◽  
Anthony Hodder ◽  
Janni Christensen ◽  
Stephen Scally ◽  
Tony Triglia ◽  
...  

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.


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