scholarly journals The Early Phase of β2m Aggregation: An Integrative Computational Study Framed on the D76N Mutant and the ΔN6 Variant

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 366 ◽  
Author(s):  
Rui J. S. Loureiro ◽  
Diogo Vila-Viçosa ◽  
Miguel Machuqueiro ◽  
Eugene I. Shakhnovich ◽  
Patrícia F. N. Faísca
Keyword(s):  
Hot Spots ◽  
Ex Vivo ◽  
Computational Study ◽  
Early Stage ◽  
Single Point ◽  
Protein Docking ◽  
Docking Simulations ◽  
C Terminus ◽  
N Terminus ◽  
Self Association

Human β2-microglobulin (b2m) protein is classically associated with dialysis-related amyloidosis (DRA). Recently, the single point mutant D76N was identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. To get insight into the early stage of the β2m aggregation mechanism, we used molecular simulations to perform an in depth comparative analysis of the dimerization phase of the D76N mutant and the ΔN6 variant, a cleaved form lacking the first six N-terminal residues, which is a major component of ex vivo amyloid plaques from DRA patients. We also provide first glimpses into the tetramerization phase of D76N at physiological pH. Results from extensive protein–protein docking simulations predict an essential role of the C- and N-terminal regions (both variants), as well as of the BC-loop (ΔN6 variant), DE-loop (both variants) and EF-loop (D76N mutant) in dimerization. The terminal regions are more relevant under acidic conditions while the BC-, DE- and EF-loops gain importance at physiological pH. Our results recapitulate experimental evidence according to which Tyr10 (A-strand), Phe30 and His31 (BC-loop), Trp60 and Phe62 (DE-loop) and Arg97 (C-terminus) act as dimerization hot-spots, and further predict the occurrence of novel residues with the ability to nucleate dimerization, namely Lys-75 (EF-loop) and Trp-95 (C-terminus). We propose that D76N tetramerization is mainly driven by the self-association of dimers via the N-terminus and DE-loop, and identify Arg3 (N-terminus), Tyr10, Phe56 (D-strand) and Trp60 as potential tetramerization hot-spots.

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

scholarly journals Insulation of Enhancer-Promoter Communication by a Gypsy Transposon Insert in the Drosophila cut Gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 Proteins

2001 ◽  
Vol 21 (14) ◽  
pp. 4807-4817 ◽  
Author(s):  
Maria Gause ◽  
Patrick Morcillo ◽  
Dale Dorsett
Keyword(s):  
Gene Activation ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Multiple Contacts ◽  
C Terminus ◽  
Insulator Activity ◽  
N Terminus ◽  
Enhancer Blocking ◽  
Self Association

ABSTRACT The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of thewhite gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-functionmod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing ofwhite. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein.

scholarly journals Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

2017 ◽  
Vol 114 (27) ◽  
pp. 7112-7117 ◽  
Author(s):  
Wan-Lin Wu ◽  
Christopher Robert Grotefend ◽  
Ming-Ting Tsai ◽  
Yi-Ling Wang ◽  
Vladimir Radic ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Dendritic Cells ◽  
Early Stage ◽  
Major Contributor ◽  
C Terminus ◽  
N Terminus ◽  
Determining Factor ◽  
Hiv 1

CCR5 (R5)-tropic, but not CXCR4 (X4)-tropic, HIV-1 is associated with primary HIV-1 infection and transmission. Recent studies have shown that IFN-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, restrict a broad range of viruses. Here, we demonstrate that an IFITM2 isoform (Δ20 IFITM2) lacking 20 amino acids at the N terminus differentially restricts X4 and R5 HIV-1. Δ20 IFITM2 suppresses replication of X4 HIV-1 strains by inhibiting their entry. High levels of Δ20 IFITM2 expression could be detected in CD4+T cells and in monocytes. Infection of X4 viruses in monocyte-derived macrophages and dendritic cells is enhanced upon depletion of IFITM2 isoforms. Furthermore, we also show that coreceptor use is the determining factor for differential HIV-1 restriction of Δ20 IFITM2. When we replace the C terminus of CCR5 with the C terminus of CXCR4, R5 viruses become more susceptible to Δ20 IFITM2-mediated restriction. In contrast to previous studies, our research reveals that neither X4 nor R5 HIV-1 is suppressed by IFITM2 and IFITM3. The multifactor gatekeeping model has been proposed to explain restriction of X4 viruses in the early stage of HIV-1 diseases. Our findings indicate that Δ20 IFITM2 may serve as a major contributor to this gatekeeping mechanism.

scholarly journals Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function

2020 ◽  
Author(s):  
Monika Seidel ◽  
Camille Rabesahala de Meritens ◽  
Louisa Johnson ◽  
Dimitris Parthimos ◽  
Mark Bannister ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Peptide Sequence ◽  
Independent Manner ◽  
Release Channel ◽  
C Terminus ◽  
Biochemical Assays ◽  
Channel Opening ◽  
N Terminus ◽  
Primary Determinant ◽  
Self Association

Abstract Aims  The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. Methods and results  We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the β8–β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8–β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8–β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8–β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8–β9 domain in channel closing. Conclusion  These results suggest that efficient N-terminus inter-subunit communication mediated by the β8–β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression.

Evolutionary adaptation of DHFR via expression of enzyme isoforms with various binding properties and dynamics behavior: a bioinformatics and computational study

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Elahe Karimi ◽  
Emran Heshmati ◽  
Khosrow Khalifeh
Keyword(s):  
Bioinformatics Analysis ◽  
Computational Study ◽  
Stable Complex ◽  
Binding Properties ◽  
C Terminus ◽  
Enzyme Isoforms ◽  
N Terminus ◽  
Mean Square Fluctuation ◽  
Simultaneous Existence ◽  
Human Dihydrofolate Reductase

Abstract We compared the binding properties and dynamics of three experimentally reviewed isoforms of human dihydrofolate reductase (DHFR). The cytoplasmic variants including isoforms1 and 2 (iso1 and iso2) are produced by alternative splicing; while the mitochondrial form is located in the mitochondria. The iso1 as the canonical sequence contains 187 residues, and iso2 differs from the iso1, where it has 1–52 residues missing at the N-terminus of canonical sequence. Here, the structural models of the iso2 and mitochondrial forms were constructed by the MODELLER program using the crystal structure of the iso1 as the template. Bioinformatics analysis on ligand-bearing structures demonstrates that mitochondrial variant forms more stable complex with ligands compared with iso1 and 2, indicating their different binding properties. The root mean square fluctuation (RMSF) data suggest that C-terminus of iso1 contains two representative highly flexible fragments, while iso2 contains a highly flexible fragment at N-terminus end. Interestingly, both ends of mitochondrial variant have a degree of rigidity. Finally, the observation of differences in structural dynamics and binding properties predicts that the simultaneous existence of enzyme isoforms is a way to increase the speed of the enzyme maneuver in response to various environmental conditions. This prediction needs to be tested experimentally.

scholarly journals Predicting stable binding modes from simulated dimers of the D76N mutant of β2-microglobulin

2021 ◽  
Author(s):  
Nuno F. B. Oliveira ◽  
Filipe E. P. Rodrigues ◽  
João N. M. Vitorino ◽  
Rui J.S. Loureiro ◽  
Patrícia FN Faísca ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Early Stage ◽  
Structural Rearrangement ◽  
Binding Mode ◽  
Protein Docking ◽  
Interfacial Region ◽  
Binding Modes ◽  
Limited Growth ◽  
C Terminus ◽  
Dynamics Simulations

The D76N mutant of the beta-2-microgobulin protein is a biologically motivated model system to study protein aggregation. There is strong experimental evidence, supported by molecular simulations, that D76N populates a highly dynamic conformation (which we originally named I2) that exposes aggregation-prone patches as a result of the detachment of the two terminal regions. Here, we use Molecular Dynamics simulations to study the stability of an ensemble of dimers of I2 generated via protein-protein docking. MM-PBSA calculations indicate that within the ensemble of investigated dimers the major contribution to interface stabilization at physiological pH comes from hydrophobic interactions between apolar residues. Our structural analysis also reveals that the interfacial region associated with the most stable binding modes are particularly rich in residues pertaining to both the N- and C-terminus, as well residues from the BC- and DE-loops. On the other hand, the less stable interfaces are stabilized by intermolecular interactions involving residues from the CD- and EF-loops. By focusing on the most stable binding modes, we used a simple geometric rule to propagate the corresponding dimer interfaces. We found that, in the absence of any kind of structural rearrangement occurring at an early stage of the oligomerization pathway, some interfaces drive a self-limited growth process, while others can be propagated indefinitely allowing the formation of long, polymerized chains. In particular, the interfacial region of the most stable binding mode reported here falls in the class of self-limited growth.

Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

1986 ◽  
Vol 56 (02) ◽  
pp. 147-150 ◽  
Author(s):  
V Pengo ◽  
M Boschello ◽  
A Marzari ◽  
M Baca ◽  
L Schivazappa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Light Transmission ◽  
Ex Vivo ◽  
Early Stage ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Dose Dependent ◽  
Plateletderived Growth Factor ◽  
Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

Preparation and properties of three analogues of [5-leucine]enkephalin, substrates for enzyme conversion studies

1985 ◽  
Vol 50 (6) ◽  
pp. 1329-1334
Author(s):  
Jaroslav Vičar ◽  
Linda Servítová ◽  
Martin Flegel ◽  
Karel Hauzer ◽  
Tomislav Barth
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Guinea Pig ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Guinea Pig Ileum ◽  
Opioid Activity ◽  
C Terminus ◽  
N Terminus ◽  
Fragment Condensation

Analogues of [5-Leu]enkephalin, prolonged by methionine on the N-terminus or, by lysine or methionine on the C-terminus were prepared by fragment condensation, purified by ion exchange chromatography or high-pressure liquid chromatography. The substances were characterised by their opioid activity in a test on guinea-pig ileum in comparison with the activity of [5-Leu]enkephalin.

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