scholarly journals The MLKL kinase-like domain dimerization is an indispensable step of mammalian MLKL activation in necroptosis signaling

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Yu Zhang ◽  
Jia Liu ◽  
Dandan Yu ◽  
Xinxin Zhu ◽  
Xiaoyan Liu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Self Assembly ◽  
Mammalian Species ◽  
Coiled Coil ◽  
Direct Consequence ◽  
Activation Process ◽  
Sequential Process ◽  
Coiled Coil Region ◽  
And Function ◽  
General Step

AbstractMLKL phosphorylation by RIP3 is the commitment step of necroptosis execution, which could induce MLKL activation featured as MLKL monomer-oligomer transition. Here, we reported that the dimerization of the MLKL kinase-like domain was the direct consequence of RIP3 triggered MLKL-phosphorylation. Two inter-dimer interfaces were found in the crystal structure of human MLKL. Mutations destroying both interfaces could prevent RIP3-induced MLKL oligomerization and necroptosis efficiently. Moreover, we confirmed MLKL self-assembly by the internal coiled-coil region is necessary for MLKL oligomerization and function. The mutations disrupting coiled-coil self-assembly repressed necroptosis, but it did not prevent RIP3-induced dimerization of the MLKL kinase-like domain. So that, MLKL activation is a sequential process, which begins with kinase-like domain dimerization, and followed by internal coiled-coil region self-assembly to form a proper MLKL oligomer. Besides human MLKL, structural and functional analysis showed the kinase-like domain dimerization was conserved among mammalian species, suggesting it is a general step of the RIP3-induced MLKL activation process.

A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function

2014 ◽  
Vol 111 (01) ◽  
pp. 79-87 ◽  
Author(s):  
Zuzana Riedelová-Reicheltová ◽  
Roman Kotlín ◽  
Jiří Suttnar ◽  
Věra Geierová ◽  
Tomáš Riedel ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Coiled Coil ◽  
Fibrin Clot ◽  
Coiled Coil Region ◽  
Platelet Aggregometry ◽  
Sds Page ◽  
Lysis Rate ◽  
Thrombotic Complications ◽  
Natural Mutation ◽  
And Function

SummaryThe aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95–98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

scholarly journals The SAM domain of mouse SAMHD1 is critical for its activation and regulation

2017 ◽  
Author(s):  
Olga Buzovetsky ◽  
Chenxiang Tang ◽  
Kirsten Knecht ◽  
Jenna M. Antonucci ◽  
Li Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bound States ◽  
Restriction Factor ◽  
Activation Process ◽  
Catalytic Core ◽  
Sam Domain ◽  
And Function ◽  
Hiv 1 ◽  
Viral Reverse Transcription

ABSTRACTHuman SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular nucleotides required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory SAM domain. In this study, we determined the first crystal structure of full-length SAMHD1 by capturing mouse SAMHD1 (mSAMHD1) structures in three different nucleotide bound states. Although mSAMHD1 and hSAMHD1 are highly similar in sequence and function, we found that mSAMHD1 possesses a more complex nucleotide-induced activation process, highlighting the regulatory role of the SAM domain. Our results provide new insights into the regulation of SAMHD1 activity, thereby will facilitate the improvement of HIV mouse models and the development of new therapies for certain cancers and autoimmune diseases.

scholarly journals A helical LC3-interacting region mediates the interaction between the retroviral restriction factor Trim5α and mammalian autophagy-related ATG8 proteins

2018 ◽  
Vol 293 (47) ◽  
pp. 18378-18386 ◽  
Author(s):  
Jeremy R. Keown ◽  
Moyra M. Black ◽  
Aaron Ferron ◽  
Melvyn Yap ◽  
Michael J. Barnett ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Life Cycle ◽  
Analytical Ultracentrifugation ◽  
Coiled Coil ◽  
Restriction Factor ◽  
Related Protein ◽  
Binding Motifs ◽  
Binding Interface ◽  
Coiled Coil Region ◽  
Tripartite Motif

The retroviral restriction factor tripartite motif–containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B). We found that Trim5α binds all mammalian ATG8s and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a β-strand motif comprising approximately six residues, LC3B binds to Trim5α via the α-helical coiled-coil region. The orientation of the structure demonstrated that LC3B could be accommodated within a Trim5α assembly that can bind the retroviral capsid. However, mutation of the binding interface does not affect retroviral restriction. Comparison of the typical linear β-strand LIR with our atypical helical LIR reveals a conservation of the presentation of residues that are required for the interaction with LC3B. This observation expands the range of LC3B-binding proteins to include helical binding motifs and demonstrates a link between Trim5α and components of the autophagosome.

scholarly journals Sharp kinking of a coiled-coil in MutS allows DNA binding and release

2019 ◽  
Vol 47 (16) ◽  
pp. 8888-8898 ◽  
Author(s):  
Doreth Bhairosing-Kok ◽  
Flora S Groothuizen ◽  
Alexander Fish ◽  
Shreya Dharadhar ◽  
Herrie H K Winterwerp ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Dna Binding ◽  
Mismatch Repair ◽  
Coiled Coil ◽  
Site Specific ◽  
Dna Mismatch ◽  
Hinge Point ◽  
Coiled Coil Region ◽  
Kink Site

Abstract DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.

scholarly journals NEMO oligomerization and its ubiquitin-binding properties

2009 ◽  
Vol 421 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Frank J. Ivins ◽  
Mark G. Montgomery ◽  
Susan J. M. Smith ◽  
Aylin C. Morris-Davies ◽  
Ian A. Taylor ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Coiled Coil ◽  
Nuclear Factor Κb ◽  
Regulatory Subunit ◽  
Activation Process ◽  
Oligomeric State ◽  
Coiled Coil Region ◽  
Ikk Complex

The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.

scholarly journals Segregation and activation of myosin IIB creates a rear in migrating cells

2008 ◽  
Vol 183 (3) ◽  
pp. 543-554 ◽  
Author(s):  
Miguel Vicente-Manzanares ◽  
Margaret A. Koach ◽  
Leanna Whitmore ◽  
Marcelo L. Lamers ◽  
Alan F. Horwitz
Keyword(s):  
Self Assembly ◽  
De Novo ◽  
Coiled Coil ◽  
Myosin Isoforms ◽  
C Terminus ◽  
Filament Bundles ◽  
A Cell ◽  
Myosin Iia ◽  
And Function ◽  
Migrating Cells

We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.

Polypeptide-engineered physical hydrogels designed from the coiled-coil region of cartilage oligomeric matrix protein for three-dimensional cell culture

2014 ◽  
Vol 2 (20) ◽  
pp. 3123-3132 ◽  
Author(s):  
Ming-Hao Yao ◽  
Jie Yang ◽  
Ming-Shuo Du ◽  
Ji-Tao Song ◽  
Yong Yu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Self Assembly ◽  
Cartilage Oligomeric Matrix Protein ◽  
Matrix Protein ◽  
Three Dimensional ◽  
Coiled Coil ◽  
The Self ◽  
Coiled Coil Region ◽  
Dimensional Cell

A class of physical hydrogels photo-cross-linked from multi-branched photopolymeriized monomers based on the self-assembly of coiled-coil polypeptide P is developed.

scholarly journals Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation

2018 ◽  
Vol 11 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Jianqun Zheng ◽  
Hao Liu ◽  
Lei Zhu ◽  
Yawen Chen ◽  
Huijie Zhao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Cell Movement ◽  
Ependymal Cells ◽  
Coiled Coil Region ◽  
Evolutionarily Conserved ◽  
Flow Through ◽  
Ependymal Cilia ◽  
And Function ◽  
Signal Transductions

Abstract Cilia are cellular protrusions containing nine microtubule (MT) doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions. Cilia require the central pair (CP) apparatus, consisting of two CP MTs covered with projections of CP proteins, for planar strokes. How the CP MTs of such ‘9 + 2’ cilia are constructed, however, remains unknown. Here we identify Spef1, an evolutionarily conserved microtubule-bundling protein, as a core CP MT regulator in mammalian cilia. Spef1 was selectively expressed in mammalian cells with 9 + 2 cilia and specifically localized along the CP. Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6, resulting in rotational beat of the ependymal cilia. Spef1, which binds to MTs through its N-terminal calponin-homologous domain, formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs. Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus. We propose that Spef1 bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.

Sex Steroids and Human Behavior: Implications for Developmental Psychopathology

CNS Spectrums ◽  
2001 ◽  
Vol 6 (1) ◽  
pp. 75-88 ◽  
Author(s):  
Gerianne M. Alexander ◽  
Bradley S. Peterson
Keyword(s):  
Sex Differences ◽  
Human Behavior ◽  
Sex Steroids ◽  
Developmental Psychopathology ◽  
Mammalian Species ◽  
Brain Structures ◽  
Postnatal Life ◽  
Brain Structure And Function ◽  
Atypical Development ◽  
And Function

AbstractIn a variety of mammalian species, prenatal androgens organize brain structures and functions that are later activated by steroid hormones in postnatal life. In humans, studies of individuals with typical and atypical development suggest that sex differences in reproductive and nonreproductive behavior derive in part from similar prenatal and postnatal steroid effects on brain development. This paper provides a summary of research investigating hormonal influences on human behavior and describes how sex differences in the prevalences and natural histories of developmental psychopathologies may be consistent with these steroid effects. An association between patterns of sexual differentiation and specific forms of psychopathology suggests novel avenues for assessing the effects of sex steroids on brain structure and function, which may in turn improve our understanding of typical and atypical development in women and men.

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