A Glance into MTHFR Deficiency at a Molecular Level

Castrense Savojardo; Giulia Babbi; Davide Baldazzi; Pier Luigi Martelli; Rita Casadio

doi:10.3390/ijms23010167

A Glance into MTHFR Deficiency at a Molecular Level

International Journal of Molecular Sciences ◽

10.3390/ijms23010167 ◽

2021 ◽

Vol 23 (1) ◽

pp. 167

Author(s):

Castrense Savojardo ◽

Giulia Babbi ◽

Davide Baldazzi ◽

Pier Luigi Martelli ◽

Rita Casadio

Keyword(s):

Protein Structure ◽

Catalytic Domain ◽

Markov Models ◽

Consensus Method ◽

Regulatory Domain ◽

Functional Protein ◽

Regulatory Domains ◽

Interaction Sites ◽

Protein Architecture ◽

Mthfr Deficiency

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.

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Multiple regulatory domains on the Byr2 protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5876 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5876-5887 ◽

Cited By ~ 59

Author(s):

H Tu ◽

M Barr ◽

D L Dong ◽

M Wigler

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Intramolecular Interaction ◽

Mitogen Activated Protein Kinase ◽

Regulatory Domain ◽

Regulatory Domains ◽

Kinase Catalytic Domain ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

G Protein Alpha Subunit

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

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SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein–protein interactions

Biochemical Journal ◽

10.1042/bj20130219 ◽

2013 ◽

Vol 454 (1) ◽

pp. 13-30 ◽

Cited By ~ 29

Author(s):

Peter H. Sugden ◽

Liam J. McGuffin ◽

Angela Clerk

Keyword(s):

Protein Interactions ◽

Catalytic Domain ◽

Oxidant Stress ◽

Focal Adhesions ◽

Germinal Centre ◽

Cell Junctions ◽

Mitogen Activated Protein Kinase ◽

Regulatory Domain ◽

Cavernous Malformations ◽

Regulatory Domains

The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)–MO25 interaction (as in the LKB1–STRADα–MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly cerebral cavernous malformations.

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A computational in silico approach to predict high-risk coding and non-coding SNPs of human PLCG1 gene

PLoS ONE ◽

10.1371/journal.pone.0260054 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0260054

Author(s):

Safayat Mahmud Khan ◽

Ar-Rafi Md. Faisal ◽

Tasnin Akter Nila ◽

Nabila Nawar Binti ◽

Md. Ismail Hosen ◽

...

Keyword(s):

Protein Structure ◽

T Cell ◽

Protein Stability ◽

In Silico ◽

Catalytic Domain ◽

Cell Lymphoma ◽

Computational Study ◽

Solvent Accessibility ◽

T Cell Lymphoma ◽

Post Translational Modification

PLCG1 gene is responsible for many T-cell lymphoma subtypes, including peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia/lymphoma along with other diseases. Missense mutations of this gene have already been found in patients of CTCL and AITL. The non-synonymous single nucleotide polymorphisms (nsSNPs) can alter the protein structure as well as its functions. In this study, probable deleterious and disease-related nsSNPs in PLCG1 were identified using SIFT, PROVEAN, PolyPhen-2, PhD-SNP, Pmut, and SNPS&GO tools. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-mutant 2.0, MUpro, Consurf, and Netsurf 2.0 server. Some SNPs were finalized for structural analysis with PyMol and BIOVIA discovery studio visualizer. Out of the 16 nsSNPs which were found to be deleterious, ten nsSNPs had an effect on protein stability, and six mutations (L411P, R355C, G493D, R1158H, A401V and L455F) were predicted to be highly conserved. Among the six highly conserved mutations, four nsSNPs (R355C, A401V, L411P and L455F) were part of the catalytic domain. L411P, L455F and G493D made significant structural change in the protein structure. Two mutations-Y210C and R1158H had post-translational modification. In the 5’ and 3’ untranslated region, three SNPs, rs139043247, rs543804707, and rs62621919 showed possible miRNA target sites and DNA binding sites. This in silico analysis has provided a structured dataset of PLCG1 gene for further in vivo researches. With the limitation of computational study, it can still prove to be an asset for the identification and treatment of multiple diseases associated with the target gene.

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Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm

Journal of Neurosurgery ◽

10.3171/jns.1992.76.1.0111 ◽

1992 ◽

Vol 76 (1) ◽

pp. 111-118 ◽

Cited By ~ 48

Author(s):

Nobutaka Minami ◽

Eiichi Tani ◽

Yukio Maeda ◽

Ikuya Yamaura ◽

Masahiro Fukami

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Catalytic Domain ◽

Limited Proteolysis ◽

Maximum Effect ◽

Regulatory Domain ◽

Calphostin C ◽

Delayed Cerebral Vasospasm ◽

Kinase C ◽

Basilar Arteries

✓ Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

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A protein architecture guided screen for modification dependent restriction endonucleases

Nucleic Acids Research ◽

10.1093/nar/gkz755 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9761-9776 ◽

Cited By ~ 5

Author(s):

Thomas Lutz ◽

Kiersten Flodman ◽

Alyssa Copelas ◽

Honorata Czapinska ◽

Megumu Mabuchi ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Proteins ◽

Dna Methyltransferase ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Restriction Endonucleases ◽

Endonuclease Activity ◽

Protein Architecture ◽

Escherichia Coli Cells ◽

Dependent Activity

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

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The LxVP and PxIxIT NFAT Motifs Bind Jointly to Overlapping Epitopes on Calcineurin’s Catalytic Domain Distant to the Regulatory Domain

Structure ◽

10.1016/j.str.2014.05.006 ◽

2014 ◽

Vol 22 (7) ◽

pp. 1016-1027 ◽

Cited By ~ 9

Author(s):

Maayan Gal ◽

Shuai Li ◽

Rafael E. Luna ◽

Koh Takeuchi ◽

Gerhard Wagner

Keyword(s):

Catalytic Domain ◽

Regulatory Domain

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Genetic Evidence for Pak1 Autoinhibition and Its Release by Cdc42

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.602 ◽

1999 ◽

Vol 19 (1) ◽

pp. 602-611 ◽

Cited By ~ 67

Author(s):

Hua Tu ◽

Mike Wigler

Keyword(s):

Protein Kinase ◽

Hybrid System ◽

Sexual Differentiation ◽

Catalytic Domain ◽

Intramolecular Interaction ◽

Intramolecular Interactions ◽

Regulatory Domain ◽

Kinase Catalytic Domain ◽

Two Hybrid ◽

Open Configuration

ABSTRACT Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.

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Bayesian hidden Markov models to identify RNA-protein interaction sites in PAR-CLIP

Biometrics ◽

10.1111/biom.12147 ◽

2014 ◽

Vol 70 (2) ◽

pp. 430-440 ◽

Cited By ~ 5

Author(s):

Jonghyun Yun ◽

Tao Wang ◽

Guanghua Xiao

Keyword(s):

Hidden Markov Models ◽

Protein Interaction ◽

Markov Models ◽

Hidden Markov ◽

Interaction Sites ◽

Protein Interaction Sites

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Binding and Transcriptional Regulation by 14-3-3 (Bmh) Proteins Requires Residues Outside of the Canonical Motif

Eukaryotic Cell ◽

10.1128/ec.00240-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 21-30 ◽

Cited By ~ 11

Author(s):

Pabitra K. Parua ◽

Elton T. Young

Keyword(s):

Regulation Of Transcription ◽

Interacting Proteins ◽

Regulatory Domain ◽

Interaction Sites ◽

Important Interaction ◽

Proposed Model ◽

Evolutionarily Conserved ◽

Regulatory Mutations ◽

Canonical Motif ◽

Transcriptional Phenotype

ABSTRACTEvolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations,ADR1calleles that alter activator-dependent gene expression. Here, we report thatADR1calleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype ofADR1cmutants.

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