scholarly journals Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

2020 ◽  
Vol 477 (5) ◽  
pp. 905-923 ◽  
Author(s):  
Sunil Singh ◽  
J. Sivaraman
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Cancer Metastasis ◽  
E3 Ligase ◽  
The Other ◽  
Hect Domain ◽  
Loop Region ◽  
The Stability

The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

scholarly journals Nutritional availability of amino acids from protein cross-linked to protect against degradation in the rumen

1984 ◽  
Vol 52 (2) ◽  
pp. 239-247 ◽  
Author(s):  
John R. Ashes ◽  
Jim L. Mangan ◽  
Gurcharn S. Sidhu
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Medicago Sativa ◽  
Terminal Ileum ◽  
Milk Protein ◽  
The Other ◽  
Molar Basis ◽  
Lactating Goats ◽  
Total Milk

1. Casein was labelled with pairs of radioactive amino acids, lysine, tyrosine and leucine, one with I4C and the other with 3H, by jugular infusion into lactating goats followed by isolation of the double-labelled casein from the milk. Total milk protein was similarly labelled by jugular infusion of [35S]cystine. U-14C-labelled fraction- 1 leaf protein was isolated from lucerne (Medicago sativa) grown in an atmosphere of 14C022. The proteins were treated withdifferent levels(333 and667 mmol/kgprotein) offormaldehyde, glutaraldehyde and glyoxal.3. Absorption from the small intestine was measured in sheep with fistulas in the abomasum and terminal ileum, using Cr-EDTA as the digesta flow marker, by introducing radioactive casein into the abomasum.4. Lysine, tyrosine and cystine became increasingly unavailable for absorption from the small intestine of sheep with increasing levels of aldehyde. At the lower level (333 mmol/kg) the proportions of the amino acids that were unavailable were 0.192, 0.051 and 0.123 respectively. At the higher level of formaldehyde (667 mmol/kg) the corresponding values were 0.335, 0.201 and 0.432 respectively. Leucine was not made unavailable with formaldehyde.5. The proportions of lysine, tyrosine and leucine that were unavailable were higher, on a molar basis, after treatment of the proteins with the dialdehydes glutaraldehyde and glyoxal than after treatment with formaldehyde. However, the extent of protein protection provided by the dialdehydes in the rumen, measured using an in vitro procedure, was lower.

scholarly journals Comparison of four methods of differential typing of isolates ofShigella sonnei

1981 ◽  
Vol 87 (3) ◽  
pp. 339-355 ◽  
Author(s):  
S. Helgason ◽  
D. C. Old
Keyword(s):  
Antibiotic Sensitivity ◽  
The Other ◽  
Drug Resistant ◽  
Typing Method ◽  
Stable Property ◽  
Discriminating Power ◽  
Sensitivity Tests ◽  
The Stability

SummaryAn epidemiological study of Sonne dysentery in Dundee during the years 1971–6 was made by examining, in respect of 1420 isolates ofShigella sonnei, the discriminating power of colicine typing, antibiogram testing, biotyping and resistotyping and the stability of the markers they provided.Colicine typing identified nine colicine types, including four not previously described. However, because types 4 and 4 var., determined bycolIb, and type U, producing no colicines, accounted for 96 % of the isolates, discrimination with colicine typing was poor. In antibiotic sensitivity tests, 13 different antibiogram patterns were noted. Less than 1 % of the isolates were sensitive to all of the eight antibiotics tested; most were multiply drug-resistant. Resistance to kanamycin, neomycin and paromomycin (KNP) was apparently due to a single resistance determinant, widely distributed in a majority (53%) of the isolates. When definitive times were chosen for reading each biotyping test, only maltose and rhamnose of the 13 ‘sugars’ tested differentiated isolates into prompt- and late-fermenting types. Though the ability to ferment rhamnose was a stable property, it discriminated only 1·5% of the minority, late-fermenting type. Resistotyping with six chemicals discriminated eight epidemiologically valid resistotypes, including three new types. However, 93 % of the isolates belonged to only three resistotypes.Analysis of the data for isolates from 286 epidemiologically distinct episodes showed that the variability of colicine and antibiogram characters, found among isolates within, respectively, 40 and 28 % of the episodes, was generally associated with loss or gain of a plasmid (‘colIb-KNP’) which determined production of colicine Ib and KNP resistance. These characters varied bothin vivoandin vitro. Variability of resistotype characters, on the other hand, was observed in only 28 (9%) episodes, 14 of which possibly represented examples of mixed or sequential infections.For accurate epidemiological tracing of strains ofSh. sonneiin a community, resistotyping, the technique showing the greatest discrimination and least variability of the four tested, should be included as the principal typing method.

The Bβ-sheet in the PAI-1 Molecule Plays an Important Role for Its Stability

2000 ◽  
Vol 83 (06) ◽  
pp. 896-901 ◽  
Author(s):  
Guang-Chao Sui ◽  
Björn Wiman
Keyword(s):  
Amino Acids ◽  
Half Life ◽  
Ph Dependence ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Reactive Center ◽  
The Stability ◽  
Β Sheet ◽  
Pai 1

SummaryWe have investigated the B β-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His219 to Tyr241 (except for Gly230 and Pro240), covering the s2B and s3B strands, and in addition His185 and His190 were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37° C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37° C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B β-sheet (Ile223 to Leu226 on s2B and Met235 to Ile237 on s3B), caused a significant decrease in stability, yielding half-lives of about 10–25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B β-sheet (Tyr221, Asp222, Tyr228 and Thr232) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His229) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B β-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A β-sheet of the molecule.

scholarly journals SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate

2018 ◽  
Author(s):  
Michael J. Roy ◽  
Sandra Winkler ◽  
Scott J. Hughes ◽  
Claire Whitworth ◽  
Michael Galant ◽  
...  
Keyword(s):  
Ternary Complex ◽  
E3 Ligase ◽  
Ternary Complexes ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Dissociation Kinetics ◽  
Intracellular Degradation ◽  
The Stability ◽  
Kinetics Of

ABSTRACTBifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

scholarly journals Regulation of poly(a)-specific ribonuclease activity by reversible lysine acetylation

2020 ◽  
Vol 295 (30) ◽  
pp. 10255-10270
Author(s):  
Eden A. Dejene ◽  
Yixuan Li ◽  
Zahra Showkatian ◽  
Hongbo Ling ◽  
Edward Seto
Keyword(s):  
Enzymatic Activity ◽  
Noncoding Rna ◽  
Critical Role ◽  
Telomere Maintenance ◽  
Post Translational Modifications ◽  
Telomere Elongation ◽  
The Stability ◽  
Human Telomerase

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3′-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3′-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.

scholarly journals Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA

2017 ◽  
Vol 114 (19) ◽  
pp. 4954-4959 ◽  
Author(s):  
Minghao Chen ◽  
Shin-ichi Asai ◽  
Shun Narai ◽  
Shusuke Nambu ◽  
Naoki Omura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzymatic Activity ◽  
Molecular Mechanism ◽  
Active Site ◽  
Thermophilic Bacteria ◽  
Oxygen Sensitive ◽  
Sulfur Protein ◽  
Combined Action

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

scholarly journals Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

2016 ◽  
Vol 113 (29) ◽  
pp. E4151-E4160 ◽  
Author(s):  
Selom K. Doamekpor ◽  
Joong-Won Lee ◽  
Nathaniel L. Hepowit ◽  
Cheng Wu ◽  
Clement Charenton ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Quality ◽  
E3 Ligase ◽  
Structure And Function ◽  
Dependent Manner ◽  
Ribosomal Subunits ◽  
Terminal Domain ◽  
Cell Extracts ◽  
And Function

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

scholarly journals Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket

2021 ◽  
Vol 22 (6) ◽  
pp. 3219
Author(s):  
Fanghua Wang ◽  
Siyu Liu ◽  
Xuejing Mao ◽  
Ruiguo Cui ◽  
Bo Yang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rational Design ◽  
Structural Information ◽  
Plant Protection ◽  
Serratia Plymuthica ◽  
Associated Bacteria ◽  
Loop Region ◽  
C Terminus ◽  
Loop Conformation

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure–function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between β9 and α9 (aa228–246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272–275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between β11 and β12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

scholarly journals UVA and UVB Photoprotective Capabilities of Topical Formulations Containing Mycosporine-like Amino Acids (MAAs) through Different Biological Effective Protection Factors (BEPFs)

Marine Drugs ◽  
2019 ◽  
Vol 17 (1) ◽  
pp. 55 ◽  
Author(s):  
Francisca de la Coba ◽  
José Aguilera ◽  
Nathalie Korbee ◽  
María de Gálvez ◽  
Enrique Herrera-Ceballos ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Tests ◽  
Antioxidant Compounds ◽  
Protection Factor ◽  
Effective Protection ◽  
Alkaline Conditions ◽  
Solar Uv ◽  
The Stability ◽  
Ph Range

The safety and stability of synthetic UV-filters and the procedures for evaluating the photoprotective capability of commercial sunscreens are under continuous review. The influence of pH and temperature stressors on the stability of certain Mycosporine-like amino acids (MAAs) isolated at high purity levels was examined. MAAs were highly stable at room temperature during 24 h at pH 4.5–8.5. At 50 °C, MAAs showed instability at pH 10.5 while at 85 °C, progressive disappearances were observed for MAAs through the studied pH range. In alkaline conditions, their degradation was much faster. Mycosporine-serinol and porphyra-334 (+shinorine) were the most stable MAAs under the conditions tested. They were included in four cosmetically stable topical sunscreens, of which the Sun Protection Factor (SPF) and other Biological Effective Protection Factors (BEPFs) were calculated. The formulation containing these MAAs showed similar SPF and UVB-BEPFs values as those of the reference sunscreen, composed of synthetic UV absorbing filters in similar percentages, while UVA-BEPFs values were slightly lower. Current in vitro data strongly suggest that MAAs, as natural and safe UV-absorbing and antioxidant compounds, have high potential for protection against the diverse harmful effects of solar UV radiation. In addition, novel complementary in vitro tests for evaluation of commercial sunscreens efficacy are proposed.

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