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Current Research in Structural Biology 2022Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single... (Review)
Review
Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.
PubMed: 36248264
DOI: 10.1016/j.crstbi.2022.09.005 -
Biochemical Society Transactions Jun 2017S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results... (Review)
Review
S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results in anchoring of soluble proteins to membrane compartments or in the trafficking of membrane proteins to different compartments. Recent work has shown that although S-acylation of some proteins may involve very weak interactions with zDHHC enzymes, a pool of zDHHC enzymes exhibit strong and specific interactions with substrates, thereby recruiting them for S-acylation. For example, the ankyrin-repeat domains of zDHHC17 and zDHHC13 interact specifically with unstructured consensus sequences present in some proteins, thus contributing to substrate specificity of these enzymes. In addition to this new information on zDHHC enzyme protein substrate specificity, recent work has also identified marked differences in selectivity of zDHHC enzymes for acyl-CoA substrates and has started to unravel the underlying molecular basis for this lipid selectivity. This review will focus on the protein and acyl-CoA selectivity of zDHHC enzymes.
Topics: Acylation; Acyltransferases; Animals; Cysteine; Humans; Membrane Proteins; Protein Interaction Domains and Motifs; Substrate Specificity
PubMed: 28620036
DOI: 10.1042/BST20160309 -
Pathology Oncology Research : POR 2022The expression of ArfGAP with SH3 domain ankyrin repeat and PH domain 1 (ASAP1) is increased in various types of cancer, showing potential as a prognostic marker. The...
The expression of ArfGAP with SH3 domain ankyrin repeat and PH domain 1 (ASAP1) is increased in various types of cancer, showing potential as a prognostic marker. The clinicopathological implications of ASAP1 expression in patients with hepatocellular carcinoma (HCC) remain unclear. We thus investigated the clinicopathological significance and prognostic effect of ASAP1 expression in HCC patients. ASAP1 expression was assessed in 149 HCC tissue samples using immunohistochemistry (IHC). The associations between ASAP1 expression and clinicopathological characteristics were analyzed. The prognostic effect of ASAP1 expression in patients with HCC was evaluated based on survival analyses and confirmed using a web-based tool. ASAP1 expression was observed in the cytoplasm of tumor cells. High ASAP1 expression was observed in 89 (59.7%) of 149 cases. High ASAP1 expression was significantly associated with male patients ( = 0.018), higher histological grade ( = 0.013), vessel invasion ( = 0.021), and higher stage ( = 0.020). High ASAP1 expression was associated with shorter overall survival (OS; = 0.041) and recurrence-free survival (RFS; = 0.008) based on Kaplan-Meier survival analyses. Web-based analysis using Kaplan-Meier (KM) plotter showed high mRNA ASAP1 expression to be associated with short OS ( = 0.001). High ASAP1 expression was associated with aggressive clinicopathological characteristics and poor clinical outcomes in patients with HCC. ASAP1 can be considered a prognostic biomarker in HCC patients.
Topics: Adaptor Proteins, Signal Transducing; Biomarkers, Tumor; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Male; Prognosis; RNA, Messenger
PubMed: 36110251
DOI: 10.3389/pore.2022.1610635 -
Toxins Mar 2020Pathogenic bacteria secrete a variety of proteins that manipulate host cell function by targeting components of the plasma membrane, cytosol, or organelles. In the last... (Review)
Review
Pathogenic bacteria secrete a variety of proteins that manipulate host cell function by targeting components of the plasma membrane, cytosol, or organelles. In the last decade, several studies identified bacterial factors acting within the nucleus on gene expression or other nuclear processes, which has led to the emergence of a new family of effectors called "nucleomodulins". In human and animal pathogens, for Gram-positive bacteria and , , , and for Gram-negative bacteria, have led to pioneering discoveries. In this review, we present these paradigms and detail various mechanisms and core elements (e.g., DNA, histones, epigenetic regulators, transcription or splicing factors, signaling proteins) targeted by nucleomodulins. We particularly focus on nucleomodulins interacting with epifactors, such as LntA of and ankyrin repeat- or tandem repeat-containing effectors of Rickettsiales, and nucleomodulins from various bacterial species acting as post-translational modification enzymes. The study of bacterial nucleomodulins not only generates important knowledge about the control of host responses by microbes but also creates new tools to decipher the dynamic regulations that occur in the nucleus. This research also has potential applications in the field of biotechnology. Finally, this raises questions about the epigenetic effects of infectious diseases.
Topics: Animals; Bacteria; Bacterial Proteins; Cell Nucleus; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Gene Expression Regulation, Bacterial; Host-Pathogen Interactions; Humans; Protein Processing, Post-Translational; Signal Transduction; Transcription, Genetic; Virulence Factors
PubMed: 32244550
DOI: 10.3390/toxins12040220 -
PLoS Computational Biology Dec 2015Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily...
Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units. We developed and applied a systematic method to consistently identify and annotate the structural repetitions over the members of the complete Ankyrin Repeat Protein Family, with increased sensitivity over previous studies. We statistically characterized the number of repeats, the folding of the repeat-arrays, their structural variations, insertions and deletions. An energetic analysis of the local frustration patterns reveal the basic features underlying fold stability and its relation to the functional binding regions. We found a strong linear correlation between the conservation of the energetic features in the repeat arrays and their sequence variations, and discuss new insights into the organization and function of these ubiquitous proteins.
Topics: Amino Acid Sequence; Ankyrin Repeat; Ankyrins; Computer Simulation; Energy Transfer; Models, Chemical; Models, Molecular; Molecular Sequence Data; Sequence Analysis, Protein
PubMed: 26691182
DOI: 10.1371/journal.pcbi.1004659 -
Pathogens (Basel, Switzerland) Aug 2022Poxviruses are double-stranded DNA viruses that infect insects and a variety of vertebrate species. The large genomes of poxviruses contain numerous genes that allow... (Review)
Review
Poxviruses are double-stranded DNA viruses that infect insects and a variety of vertebrate species. The large genomes of poxviruses contain numerous genes that allow these viruses to successfully establish infection, including those that help evade the host immune response and prevent cell death. Ankyrin-repeat (ANKR)/F-box proteins are almost exclusively found in poxviruses, and they function as substrate adapters for Skp1-Cullin-1-F-box protein (SCF) multi-subunit E3 ubiquitin (Ub)-ligases. In this regard, they use their C-terminal F-box domain to bind Skp1, Cullin-1, and Roc1 to recruit cellular E2 enzymes to facilitate the ubiquitylation, and subsequent proteasomal degradation, of proteins bound to their N-terminal ANKRs. However, these proteins do not just function as substrate adapters as they also have Ub-independent activities. In this review, we examine both Ub-dependent and -independent activities of ANKR/F-box proteins and discuss how poxviruses use these proteins to counteract the host innate immune response, uncoat their genome, replicate, block cell death, and influence transcription. Finally, we consider important outstanding questions that need to be answered in order to better understand the function of this versatile protein family.
PubMed: 36014996
DOI: 10.3390/pathogens11080875 -
BMB Reports Aug 2020ANKHD1 (ankyrin repeat and KH domain containing 1) is a large protein characterized by the presence of multiple ankyrin repeats and a K-homology domain. Ankyrin repeat... (Review)
Review
ANKHD1 (ankyrin repeat and KH domain containing 1) is a large protein characterized by the presence of multiple ankyrin repeats and a K-homology domain. Ankyrin repeat domains consist of widely existing protein motifs in nature, they mediate protein-protein interactions and regulate fundamental biological processes, while the KH domain binds to RNA or ssDNA and is associated with transcriptional and translational regulation. In recent years, studies containing relevant information on ANKHD1 in cancer biology and its clinical relevance, as well as the increasing complexity of signaling networks in which this protein acts, have been reported. Among the signaling pathways of interest in oncology regulated by ANKHD1 are Hippo signaling, JAK/STAT, and STMN1. The scope of the present review is to survey the current knowledge and highlight future perspectives for ANKHD1 in the malignant phenotype of cancer cells, exploring biological, functional, and clinical reports of this protein in cancer. [BMB Reports 2020; 53(8): 413-418].
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Binding Sites; Biological Phenomena; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 1; Neoplasms; Protein Domains; RNA; RNA-Binding Proteins; STAT Transcription Factors; Signal Transduction; Stathmin; Transcription Factors; YAP-Signaling Proteins
PubMed: 32635985
DOI: 10.5483/BMBRep.2020.53.8.087 -
The Journal of Biological Chemistry Jan 2022Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a...
Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.
Topics: Amino Acid Sequence; Antiviral Agents; COVID-19; Designed Ankyrin Repeat Proteins; Drug Development; Drug Stability; SARS-CoV-2; Sequence Alignment; Temperature; COVID-19 Drug Treatment
PubMed: 34793836
DOI: 10.1016/j.jbc.2021.101403 -
Biochimica Et Biophysica Acta.... Jun 2023The objective of this study was to explore the role of ferroptosis in the formation of calcium oxalate (CaOx) kidney stones and the regulatory mechanism of the ankyrin...
The objective of this study was to explore the role of ferroptosis in the formation of calcium oxalate (CaOx) kidney stones and the regulatory mechanism of the ankyrin repeat domain 1 (ANKRD1) gene. The study found that the Nrf2/HO-1 and p53/SLC7A11 signaling pathways were activated in the kidney stone model group, and the expression of the ferroptosis marker proteins SLC7A11 and GPX4 was significantly reduced, while the expression of ACSL4 was significantly increased. The expression of the iron transport-related proteins CP and TF increased significantly, and Fe accumulated in the cell. The expression of HMGB1 increased significantly. In addition, the level of intracellular oxidative stress was increased. The gene with the most significant difference caused by CaOx crystals in HK-2 cells was ANKRD1. Silencing or overexpression of ANKRD1 by lentiviral infection technology regulated the expression of the p53/SLC7A11 signaling pathway, which regulated the ferroptosis induced by CaOx crystals. In conclusion, CaOx crystals can mediate ferroptosis through the Nrf2/HO-1 and p53/SLC7A11 pathways, thereby weakening the resistance of HK-2 cells to oxidative stress and other unfavorable factors, enhancing cell damage, and increasing crystal adhesion and CaOx crystal deposition in the kidney. ANKRD1 participates in the formation and development of CaOx kidney stones by activating ferroptosis mediated by the p53/SLC7A11 pathway.
Topics: Humans; Calcium Oxalate; Ferroptosis; NF-E2-Related Factor 2; Tumor Suppressor Protein p53; Kidney Calculi; Muscle Proteins; Nuclear Proteins; Repressor Proteins
PubMed: 36907445
DOI: 10.1016/j.bbamcr.2023.119452 -
Nature Communications Sep 2022Over 70% of vascular flowering plants engage in endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. VAPYRIN (VPY) is a plant protein that is required for...
Over 70% of vascular flowering plants engage in endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. VAPYRIN (VPY) is a plant protein that is required for intracellular accommodation of AM fungi but how it functions is still unclear. VPY has a large ankyrin repeat domain with potential for interactions with multiple proteins. Here we show that overexpression of the ankyrin repeat domain results in a vpy-like phenotype, consistent with the sequestration of interacting proteins. We identify distinct ankyrin repeats that are essential for intracellular accommodation of arbuscules and reveal that VPY functions in both the cytoplasm and nucleus. VPY interacts with two kinases, including DOES NOT MAKE INFECTIONS3 (DMI3), a nuclear-localized symbiosis signaling kinase. Overexpression of VPY in a symbiosis-attenuated genetic background results in a dmi3 -like phenotype suggesting that VPY negatively influences DMI3 function. Overall, the data indicate a requirement for VPY in the nucleus and cytoplasm where it may coordinate signaling and cellular accommodation processes.
Topics: Ankyrin Repeat; Medicago truncatula; Mycorrhizae; Plant Proteins; Plant Roots; Symbiosis
PubMed: 36064777
DOI: 10.1038/s41467-022-32124-3