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Variants in the KCNQ1 gene, encoding the α-subunit of the slow component of delayed rectifier K+ channel Kv7.1, cause long QT syndrome (LQTS) type 1. The location of variants may be one of the factors in determining prognosis. However, detailed genotype-phenotype relationships associated with C-terminus variants remain unelucidated. We investigated the clinical characteristics and variant-specific arrhythmic risks in patients with LQTS carrying Kv7.1 C-terminus variants. The study comprises 202 consecutive patients with LQTS (98 probands and 104 family members) who carry a rare heterozygous variant in the Kv7.1 C-terminus. Their clinical characteristics and arrhythmic events were investigated. We identified 36 unique C-terminus variants (25 missense and 11 non-missense). The p.R366W variant was identified in 8 families, and p.T587M was identified in 21 families in large numbers from northwestern Japan. As for the location of the variant, we found that the variants in highly conserved regions and nonhelical domains were associated with longer QTc intervals compared with the variants in other regions. Both p.R366W and p.T587M variants are located in the highly conserved and functionally pivotal regions close to helices A and D, which are associated with calmodulin binding and channel assembly (tetramerization), respectively. The probands carrying p.T587M and p.R366W variants had worse arrhythmia outcomes compared with those with other C-terminus variants. The haplotype analysis of p.T587M families was suggestive of a founder effect. The arrhythmic risk of C-terminus variants in Kv7.1 in LQTS is not homogeneous, and locations of variants can be a determining factor for prognosis. [Display omitted] [ABSTRACT FROM AUTHOR]

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24–30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27...E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability. [ABSTRACT FROM AUTHOR]

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs. • Ca2+ influx is necessary for the phosphorylation of ERK1/2 via PIEZO1 activation in BMSCs. • The C-terminus containing the R-Ras-binding region of PIEZO1 plays a role in Ca2+ influx. • PIEZO1 activates the ERK1/2 signaling pathway which requires the R-Ras-binding domain at the C-terminus of PIEZO1. [ABSTRACT FROM AUTHOR]

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development. [ABSTRACT FROM AUTHOR]

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342–Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321–I327), and the second one encompassed a stretch of amino acid residues I348–Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of oligomerization remain relatively unexplored. Herein, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives receptor homo-oligomerization. The formation of A2AR oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A2AR and other GPCRs reconstituted in vitro for biophysical studies.

All proteins have a carboxyl terminus, and we previously summarized eight mutations in binding and trafficking sequence determinants in the C-terminus that, when disrupted, cause human diseases. These sequence elements for binding and trafficking sites, as well as post-translational modifications (PTMs), are called minimotifs or short linear motifs. We wanted to determine how frequently mutations in minimotifs in the C-terminus cause disease. We searched specifically for PTMs because mutation of a modified amino acid almost always changes the chemistry of the side chain and can be interpreted as loss-of-function. We analyzed data from ClinVar for disease variants, Minimotif Miner and the C-terminome for PTMs, and RefSeq for protein sequences, yielding 20 such potential disease-causing variants. After additional screening, they include six with a previously reported PTM disruption mechanism and nine with new hypotheses for mutated minimotifs in C-termini that may cause disease. These mutations were generally for different genes, with four different PTM types and several different diseases. Our study helps to identify new molecular mechanisms for nine separate variants that cause disease, and this type of analysis could be extended as databases grow and to binding and trafficking motifs. We conclude that mutated motifs in C-termini are an infrequent cause of disease. [ABSTRACT FROM AUTHOR]

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5–3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in ; the value of kcat has not changed. When T250H is replaced, an increase in is observed, kcat decreases from 20 to 13 s–1. The replacement of K368H led to a slight increase in , kcat decreased from 20 to 6 s–1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in and . A bigger effect was observed for —the difference was more than 10 times. For mutant SauFDH with insertions kcat significantly reduced to 4 s–1. Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH. [ABSTRACT FROM AUTHOR]

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24–30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

The small GTPase Ran is the main regulator of the nucleo-cytoplasmic import and export through the nuclear pore complex. It functions as a molecular switch cycling between the GDP-bound inactive and GTP-bound active state. It consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. Crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD). Whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, starting from the crystal structure of Ran in both GDP- and GTP-bound forms we show how rigid the C-terminal region in the inactive structure is during molecular dynamics (MD) simulations. Furthermore, we show how MD simulations of the active form are incapable of mapping the open conformations of the C-terminus. By using the MDeNM (Molecular Dynamics with excited Normal Modes) method, we were able to widely map the conformational surface of the C-terminus of Ran in the active GTP-bound form, which allows us to envisage how it can embrace RanBDs.

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as “fishing lines”, expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development.

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342–Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321–I327), and the second one encompassed a stretch of amino acid residues I348–Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD.

Abstract Background 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. Results A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. Conclusions The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used. [ABSTRACT FROM AUTHOR]

All proteins have a carboxyl terminus, and we previously summarized eight mutations in binding and trafficking sequence determinants in the C-terminus that, when disrupted, cause human diseases. These sequence elements for binding and trafficking sites, as well as post-translational modifications (PTMs), are called minimotifs or short linear motifs. We wanted to determine how frequently mutations in minimotifs in the C-terminus cause disease. We searched specifically for PTMs because mutation of a modified amino acid almost always changes the chemistry of the side chain and can be interpreted as loss-of-function. We analyzed data from ClinVar for disease variants, Minimotif Miner and the C-terminome for PTMs, and RefSeq for protein sequences, yielding 20 such potential disease-causing variants. After additional screening, they include six with a previously reported PTM disruption mechanism and nine with new hypotheses for mutated minimotifs in C-termini that may cause disease. These mutations were generally for different genes, with four different PTM types and several different diseases. Our study helps to identify new molecular mechanisms for nine separate variants that cause disease, and this type of analysis could be extended as databases grow and to binding and trafficking motifs. We conclude that mutated motifs in C-termini are an infrequent cause of disease.

Abstract The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.

Abstract Background Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported. Results In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs’ functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins. Conclusions Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs’ molecular roles in mediating other regulators’ transport to coordinate growth and development of various cotton tissues.

Main conclusion: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context. [ABSTRACT FROM AUTHOR]

Background: 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. Results: A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. Conclusions: The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable. [ABSTRACT FROM AUTHOR]

The transcription factor p73 is a structural and functional homolog of TP53, the most famous and frequently mutated tumor-suppressor gene. The TP73 gene can synthesize an overwhelming number of isoforms via splicing events in 5′ and 3′ ends and alternative promoter usage. Although it originally came into the spotlight due to the potential of several of these isoforms to mimic p53 functions, it is now clear that TP73 has its own unique identity as a master regulator of multifaceted processes in embryonic development, tissue homeostasis, and cancer. This remarkable functional pleiotropy is supported by a high degree of mechanistic heterogeneity, which extends far-beyond the typical mode of action by transactivation and largely relies on the ability of p73 isoforms to form protein–protein interactions (PPIs) with a variety of nuclear and cytoplasmic proteins. Importantly, each p73 isoform carries a unique combination of functional domains and residues that facilitates the establishment of PPIs in a highly selective manner. Herein, we summarize the expanding functional repertoire of TP73 in physiological and oncogenic processes. We emphasize how TP73’s ability to control neurodevelopment and neurodifferentiation is co-opted in cancer cells toward neoneurogenesis, an emerging cancer hallmark, whereby tumors promote their own innervation. By further exploring the canonical and non-canonical mechanistic patterns of p73, we apprehend its functional diversity as the result of a sophisticated and coordinated interplay of: (a) the type of p73 isoforms (b) the presence of p73 interaction partners in the cell milieu, and (c) the architecture of target gene promoters. We suppose that dysregulation of one or more of these parameters in tumors may lead to cancer initiation and progression by reactivating p73 isoforms and/or p73-regulated differentiation programs thereof in a spatiotemporally inappropriate manner. A thorough understanding of the mechanisms supporting p73 functional diversity is of paramount importance for the efficient and precise p73 targeting not only in cancer, but also in other pathological conditions where TP73 dysregulation is causally involved.

[Display omitted] • Interleukin receptor-associated kinase 2 (IRAK2) possesses an evolutionary conserved C-terminus. • The last 55 amino acids lack any known functional domain. • The C-terminus deletion mutant IRAK2Δ55 was still capable to bind to the E3-ubiquitin ligase TRAF6. • Without the last 55 residues, IRAK-dependent (auto-) ubiquitination of TRAF6 was impaired. • Accordingly, IRAK2Δ55 was hypofunctional in TLR signaling. Macrophages are fundamental for initiation, maintenance, and resolution of inflammation. They can be activated by 'Toll-like receptor' (TLR) engagement, which initiates critical pathways to fight infections. 'Interleukin receptor-associated kinase 2′ (IRAK2) is part of the membrane-proximal Myddosome formed at IL-1R/TLRs, but utility and regulation of IRAK2 within is not completely understood. In this study, we addressed the importance of the evolutionary conserved extreme C-terminus of IRAK2 in TLR signaling. The last 55 amino acids lack any known functional domain. The C-terminus deletion mutant IRAK2Δ55 was hypofunctional and disabled to conduct TLR4-inducible NF-κB and ERK2 activation. Accordingly, it could neither fully support subsequent CD40 cell surface expression nor IL-6 and nitric oxide release. Interestingly, IRAK2Δ55 was still capable to bind to 'tumor necrosis factor receptor-associated factor 6′ (TRAF6), which is requisite to activate TRAF6 as an E3-ubiquitin ligase for further downstream signaling. However, IRAK-dependent auto-ubiquitination of TRAF6 was impaired, when IRAK2Δ55 was bound. Thus, the conserved last 55 amino acids enable IRAK2 to sustain an optimal TLR response. This knowledge might spark ideas how overshooting inflammatory responses could be modified without blocking the entire immune response. [ABSTRACT FROM AUTHOR]

Pannexin 1 (Panx1) has been implicated in a plethora of physiological and pathophysiological processes. It is one of the major ATP release channels in many cell types. Extracellular ATP, activates purinergic P2X and P2Y receptors, triggering several signaling cascades. A disease-associated mutation, Arg-217-His (R217H) in the 3rd transmembrane domain of Panx1 attenuates channel functions through an unknown mechanism. Since carboxyl terminus (CT) gates the channel, we hypothesized that R217 interacts with the CT, and this interaction is required for optimum channel activities. R217H mutation though reduced the currents in the full-length channel, did not affect CT-truncated Panx1-Δ386. Also, compared to the wild-type, Panx1-R217H expressing cells showed lesser cell death when activated through P2X7 receptor. However, cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells were similar. The mutation is ineffective unless the channel has an intact CT. Based on our results we propose that R217H mutation perturbs the conformational flexibility of CT, leading to channel dysfunction. • Disease-associated mutant Pannexin 1 (Panx1), R217H exhibited reduced currents. • R217H when introduced in the C-terminus (CT)-truncated Panx1-Δ386, had no effect. • Cells expressing Panx1-R217H showed lesser death than wild-type Panx1. • Cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells are similar. • Possibly, the interaction between R-217 and CT is affected by the mutation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Yeast Na+/H+ antiporters differ in cation selectivity for Li+, Na+ and/or K+. • The hydrophilic C-terminus influences the transport of particular cations. • Composition of the conserved C3 domain impacts the ability to transport K+. • Physiological functions of yeast Na+/H+ antiporters depend on their C-termini. Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii , the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled Zr Sod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of Zr Sod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of Zr Sod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in Sc Nha1, the replacement of three positively charged arginine residues 539–541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts. [ABSTRACT FROM AUTHOR]

Several water-soluble variants of the human mu opioid receptor (wsMORs) have been designed and expressed, which enables the detection of opioids in the nM to pM range using biosensing platforms. The tools previously developed allowed us to investigate MOR and G-protein interactions in a lipid free system to demonstrate that the lipid bilayer might not be essential for the G-protein recognition and binding. In this study, we are able to investigate G-protein interactions with MOR by using graphene enabled technology, in a lipid free system, with a high sensitivity in a real time manner. A new wsMOR with the native C-terminus was designed, expressed and then immobilized on the surfaces of scalable graphene field effect transistor (GFET)-based biosensors, enabling the recording of wsMOR/G-protein interaction with an electronic readout. G-protein only interacts with the wsMOR in the presence of the native MOR C-terminus with a $\text{K}_{A}$ of 32.3± 11.1 pM. The electronic readout of such interaction is highly reproducible with little variance across 50 devices in one biosensor array. For devices with receptors that do not have the native C-terminus, no significant electronic response was observed in the presence of G-protein, indicating an absence of interaction. These findings reveal that lipid environment is not essential for the G-protein interaction with MOR, however, the C-terminus of MOR is essential for G-protein recognition and high affinity binding. A system to detect MOR-G protein interaction is developed. wsMOR-G2_Cter provides a novel tool to investigate the role of C terminus in the signaling pathway. [ABSTRACT FROM AUTHOR]

Cargo transport within cells is essential to healthy cells, which requires microtubules-based motors, including kinesin. The C-terminal tails (E-hooks) of alpha and beta tubulins of microtubules have been proven to play important roles in interactions between the kinesins and tubulins. Here, we implemented multi-scale computational methods in E-hook-related analyses, including flexibility investigations of E-hooks, binding force calculations at binding interfaces between kinesin and tubulins, electrostatic potential calculations on the surface of kinesin and tubulins. Our results show that E-hooks have several functions during the binding process: E-hooks utilize their own high flexibilities to increase the chances of reaching a kinesin; E-hooks help tubulins to be more attractive to kinesin. Besides, we also observed the differences between alpha and beta tubulins: beta tubulin shows a higher flexibility than alpha tubulin; beta tubulin generates stronger attractive forces (about twice the strengths) to kinesin at different distances, no matter with E-hooks in the structure or not. Those facts may indicate that compared to alpha tubulin, beta tubulin contributes more to attracting and catching a kinesin to microtubule. Overall, this work sheds the light on microtubule studies, which will also benefit the treatments of neurodegenerative diseases, cancer treatments, and preventions in the future.

The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the protein’s transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology.

Background: Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported. Results: In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs' functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins. Conclusions: Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs' molecular roles in mediating other regulators' transport to coordinate growth and development of various cotton tissues. [ABSTRACT FROM AUTHOR]

The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the protein's transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology. [ABSTRACT FROM AUTHOR]

Extracellular adenine nucleotides play important roles in cell–cell communication and tissue homeostasis. High concentrations of extracellular ATP released by dying cells are sensed as a danger signal by the P2X7 receptor, a non-specific cation channel. Studies in P2X7 knockout mice and numerous disease models have demonstrated an important role of this receptor in inflammatory processes. P2X7 activation has been shown to induce a variety of cellular responses that are not usually associated with ion channel function, for example changes in the plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis. In contrast to all other P2X family members, the P2X7 receptor contains a long intracellular C-terminus that constitutes 40% of the whole protein and is considered essential for most of these effects. So far, over 50 different proteins have been identified to physically interact with the P2X7 receptor. However, few of these interactions have been confirmed in independent studies and for the majority of these proteins, the interaction domains and the physiological consequences of the interactions are only poorly described. Also, while the structure of the P2X7 extracellular domain has recently been resolved, information about the organization and structure of its C-terminal tail remains elusive. After shortly describing the structure and assembly of the P2X7 receptor, this review gives an update of the identified or proposed interaction domains within the P2X7 C-terminus, describes signaling pathways in which this receptor has been involved, and provides an overlook of the identified interaction partners.

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu2+ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg2+ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl2 strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2–3-fold lower than that in the MgCl2-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.

Extracellular adenine nucleotides play important roles in cell–cell communication and tissue homeostasis. High concentrations of extracellular ATP released by dying cells are sensed as a danger signal by the P2X7 receptor, a non-specific cation channel. Studies in P2X7 knockout mice and numerous disease models have demonstrated an important role of this receptor in inflammatory processes. P2X7 activation has been shown to induce a variety of cellular responses that are not usually associated with ion channel function, for example changes in the plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis. In contrast to all other P2X family members, the P2X7 receptor contains a long intracellular C-terminus that constitutes 40% of the whole protein and is considered essential for most of these effects. So far, over 50 different proteins have been identified to physically interact with the P2X7 receptor. However, few of these interactions have been confirmed in independent studies and for the majority of these proteins, the interaction domains and the physiological consequences of the interactions are only poorly described. Also, while the structure of the P2X7 extracellular domain has recently been resolved, information about the organization and structure of its C-terminal tail remains elusive. After shortly describing the structure and assembly of the P2X7 receptor, this review gives an update of the identified or proposed interaction domains within the P2X7 C-terminus, describes signaling pathways in which this receptor has been involved, and provides an overlook of the identified interaction partners. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Comparison of LB400, KT2440 and ADP1 intradiol dioxygenase activities. • Protein engineering to increase intradiol dioxygenase activity (Kcat). • Elimination of C-terminal from ClcA and CatA by manipulation of the structural gene. Abstract Intradiol dioxygenases (EC 1.13.11.1) are bacterial enzymes that catalyze the ring cleavage of catechols which is a central step in the aerobic degradation of aromatic compounds. Some members of this enzyme group have a C-terminus which is 4–5% longer (an additional 13–18 amino acids) compared to the majority of known sequences. The longer C-terminus itself is not highly conserved and appears to be poorly integrated in the protein structural models developed for representative intradiol dioxygenases. Using a protein engineering approach variant intradiol dioxygenases were produced by truncating the C-terminus to a size comparable to the shorter versions of the enzyme. Three enzymes were selected and were originally described from the model organisms; Burkholderia xenovorans LB400, Pseudomonas putida KT2440 and Acinetobacter baylyi ADP1. The activity of the truncated enzymes were compared to the unmodified enzymes which revealed that truncation of the C-terminus could alter the enzyme activity; increasing the LB400 enzyme activity by as much as five fold, but reducing the activity of the intradiol dioxygenases from KT2440 and ADP1. The difference in effect is explained by the presence of a greater number of amino acid residues that can contribute to forming stable protein structures in the KT2440 and ADP1 enzymes. It is hypothesized that C-terminal truncation could in some cases provide a useful strategy for increasing intradiol dioxygenase activity for biotechnological production of muconic and adipic acids. [ABSTRACT FROM AUTHOR]

All proteins end with a carboxyl terminus that has unique biophysical properties and is often disordered. Although there are examples of important C-termini functions, a more global role for the C-terminus is not yet established. In this review, we summarize research on C-termini, a unique region in proteins that cells exploit. Alternative splicing and proteolysis increase the diversity of proteins and peptides in cells with unique C-termini. The C-termini of proteins contain minimotifs, short peptides with an encoded function generally characterized as binding, posttranslational modifications, and trafficking. Many of these activities are specific to minimotifs on the C-terminus. Approximately 13% of C-termini in the human proteome have a known minimotif, and the majority, if not all of the remaining termini have conserved motifs inferring a function that remains to be discovered. C-termini, their predictions, and their functions are collated in the C-terminome, Proteus, and Terminus Oriented Protein Function INferred Database (TopFIND) database/web systems. Many C-termini are well conserved, and some have a known role in health and disease. We envision that this summary of C-termini will guide future investigation of their biochemical and physiological significance. [ABSTRACT FROM AUTHOR]

Anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum (ER) through the Golgi apparatus represents a crucial checkpoint to control the amount of the receptors at the functional destination and the strength of receptor activation‐elicited cellular responses. However, as compared with extensively studied internalization and recycling processes, the molecular mechanisms of cell surface trafficking of GPCRs are relatively less defined. Here, we will review the current advances in understanding the ER‐Golgi‐cell surface transport of GPCRs and use angiotensin II type 1 receptor as a representative GPCR to discuss emerging roles of receptor‐interacting proteins and specific motifs embedded within the receptors in controlling the forward traffic of GPCRs along the biosynthetic pathway. As compared with internalization and recycling, anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum through the Golgi apparatus remains poorly understood. Here, we will review the current understanding of the cell surface trafficking of the GPCR superfamily and use AT1R as an example to discuss emerging roles of receptor‐interacting proteins and specific motifs. [ABSTRACT FROM AUTHOR]

Background: Adenocarcinoma of the pancreas is one of the most aggressive tumor diseases affecting the human body. The oncogenic potential of pancreatic cancer is mainly characterized by extremely rapid growth triggered by the activation of oncogenic signaling cascades, which suggests a change in the regulation of important transcription factors. Amongst others, NFAT transcription factors are assumed to play a central role in the carcinogenesis of pancreatic cancer. Recent research has shown the importance of the transcription factor Sp1 in the transcriptional activity of NFATc2 in pancreatic cancer. However, the role of the interaction between these two binding partners remains unclear. The current study investigated the role of Sp1 proteins in the expression of NFATc2 target genes and identified new target genes and their function in cells. A further objective was the domain of the Sp1 protein that mediates interaction with NFATc2. The involvement of Sp1 proteins in NFATc2 target genes was shown by means of a gene expression profile analysis, and the results were confirmed by quantitative RT-PCR. The functional impact of this interaction was shown in a thymidine incorporation assay. A second objective was the physical interaction between NFATc2 and different Sp1 deletion mutants that was investigated by means of immunoprecipitation. Results: In pancreatic cancer, the proto-oncogene c-Fos, the tumor necrosis factor TNF-alpha, and the adhesion molecule integrin beta-3 are target genes of the interaction between Sp1 and NFATc2. Loss of just one transcription factor inhibits oncogenic complex formation and expression of cell cycle-regulating genes, thus verifiably decreasing the carcinogenic effect. The current study also showed the interaction between the transcription factor NFATc2 and the N-terminal domain of Sp1 in pancreatic cancer cells. Sp1 increases the activity of NFATc2 in the NFAT-responsive promoter. Conclusions: The regulation of gene promotors during transcription is a rather complex process because of the involvement of many proteins that – as transcription factors or co-factors – regulate promotor activity as required and control cell function. NFATc2 and Sp1 seem to play a key role in the progression of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A2AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A2AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for Gα protein-associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Gα (i.e., Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the Gα protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.

Protein C-termini containing valuable biological information plays a vital role in various physiological processes, such as protein localization, protein recognition, and signal transduction in organisms. However, C-terminal peptide identification is still challenging due to their low abundance and similar physicochemical properties to other digested peptides. Herein, we developed a simple and mild strategy for the enrichment of C-terminal peptides that incorporates selectively 2-pyridinecarbaldehyde (2-PCA) derivatization of α-amine with negative enrichment by NHS resin. Two synthesized peptides were utilized to evaluate the efficiency of 2-PCA derivatization and optimize the coupling conditions of NHS resin. The feasibility of the method was further validated by enriching the C-terminus of the bovine serum albumin (BSA). Finally, this method was successfully applied to the C-terminus analysis of mouse brain tissue, identifying 404 protein C-termini with physicochemical properties unbiasedly. Additionally, the GO and KEGG analyses revealed that these identified proteins are crucial for proper brain function. In summary, our proposed method is effective and has the potential to facilitate comprehensive C-terminal analysis of proteins. Effective enrichment methods are essential for the identification of the proteins C-terminus. In this study, a mild and simple method for negative C-terminal enrichment combined with site-specific derivatization was developed. The enrichment process was simplified and minimized sample loss simultaneously, using 2-PCA derivatization which has high α-amino specificity. Up to 346C-terminal proteins were identified in mouse brain tissue unbiasedly and reliably. This approach has the potential to facilitate comprehensive analysis of protein C-termini in a variety of biological contexts. [Display omitted] • Site specificity reaction with 2-PCA was applied in α-amino blocking of digested peptides by LysC. • Internal peptides with ε-NH2 were captured by NHS-resin leading to separated C-terminal peptides in aqueous easily for enrichment. • C-terminal peptides of mouse brain tissue were successfully enriched and analyzed by LC-MS/MS. [ABSTRACT FROM AUTHOR]