Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MSn (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.

Download Full-text

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽

10.3390/cancers13133317 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3317

Author(s):

Eric Moeglin ◽

Dominique Desplancq ◽

Audrey Stoessel ◽

Christian Massute ◽

Jeremy Ranniger ◽

...

Keyword(s):

Cancer Cells ◽

Mammalian Cells ◽

Chromatin Modification ◽

Replication Stress ◽

Living Cells ◽

Single Step ◽

Dna Double Strand Breaks ◽

Drug Induced ◽

Histone H2ax ◽

C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Download Full-text

Improving Tandem Mass Spectra Analysis with Hierarchical Learning

Proceedings of the Twenty-Ninth International Joint Conference on Artificial Intelligence ◽

10.24963/ijcai.2020/599 ◽

2020 ◽

Author(s):

Zhengcong Fei

Keyword(s):

Peptide Identification ◽

Complete Sequence ◽

Peptide Sequence ◽

Tandem Mass ◽

Spectra Analysis ◽

Hierarchical Learning ◽

Tandem Mass Spectra ◽

Multi Stage ◽

Public Datasets ◽

Fine Tune

Tandem mass spectrometry is the most widely used technology to identify proteins in a complex biological sample, which produces a large number of spectra representative of protein subsequences named peptide. In this paper, we propose a hierarchical multi-stage framework, referred as DeepTag, to identify the peptide sequence for each given spectrum. Compared with the traditional one-stage generation, our sequencing model starts the inference with a selected high-confidence guiding tag and provides the complete sequence based on this guiding tag. Besides, we introduce a cross-modality refining module to asist the decoder focus on effective peaks and fine-tune with a reinforcement learning technique. Experiments on different public datasets demonstrate that our method achieves a new state-of-the-art performance in peptide identification task, leading to a marked improvement in terms of both precision and recall.

Download Full-text

Yeast-Based Directed-Evolution For High-Throughput Structural Stabilization of G Protein-Coupled Receptors (GPCRs)

10.21203/rs.3.rs-1066896/v1 ◽

2021 ◽

Author(s):

May Meltzer ◽

Zvagelsky Tatiana ◽

Niv Papo ◽

Stanislav Engel

Keyword(s):

G Protein ◽

Resistant Cell ◽

Single Step ◽

G Protein Coupled Receptors ◽

Structural Basis ◽

Diffusion Method ◽

Structural Stabilization ◽

C Terminus ◽

Screening Platform ◽

G Protein Coupled

Abstract The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we presented a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The detergent-resistant cell wall of yeast provides a unique compartmentalization opportunity to physically link the receptor phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method offers important insights into the structural basis of GPCR stability, questioning the inherent instability of the GPCR scaffold, and revealing the potential role of the C-terminus in receptor stabilization.

Download Full-text

Structural basis for UFM1 transfer from UBA5 to UFC1

Nature Communications ◽

10.1038/s41467-021-25994-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Manoj Kumar ◽

Prasanth Padala ◽

Jamal Fahoum ◽

Fouad Hassouna ◽

Tomer Tsaban ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Structural Basis ◽

Adenylation Domain ◽

Post Translational Modification ◽

Target Proteins ◽

Cellular Processes ◽

Enzyme Cascade ◽

Linear Sequence ◽

C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

Download Full-text

PP2A Methylation is Necessary for Broad Stress Tolerance

10.21203/rs.3.rs-709547/v1 ◽

2021 ◽

Author(s):

Maria T. Creighton ◽

Dugassa Nemie-Feyissa ◽

Nabeela Zaman ◽

Sverre S. Johansen ◽

Hege Dysjaland ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Tolerance ◽

Dry Weight ◽

Sodium Concentration ◽

Post Translational Modification ◽

Iron Starvation ◽

Methyl Transferase ◽

High Concentrations ◽

Fe Homeostasis ◽

Rock Wool

Abstract Background: LEUCINE CARBOXYL METHYL TRANSFERASE 1 (LCMT1) transfers a methyl group from the methyl donor S-adenosylmethionine (SAM) to the catalytic subunit of PROTEIN PHOSPHATASE 2A (PP2A). This post-translational modification of PP2A is manifested throughout eukaryotes from yeast to plants and animals. Although highly conserved, the importance of the methylation is poorly understood. Since Arabidopsis plants with knocked out LCMT1 grow and develop fairly normally, we decided to search for conditions that may reveal the benefits of this regulation. We compared the effects of various stressful conditions on Arabidopsis wild type (WT) and a lcmt1 mutant possessing only non-methylated PP2A. Results: Seedlings were grown in Petri dishes for 5-12 days, or in rock wool and soil for up to 7 weeks. A significant increase in sodium concentration was found for lcmt1 relative to WT, but this was not linked with stressful conditions. Plants were exposed to variable levels of the chelator EDTA, iron, zinc, aluminium, heat, and hydrogen peroxide. The lcmt1 mutant was clearly more sensitive than WT to all the various stresses, as demonstrated by effects on seedling root growth and on shoots of rosette stage plants on rock wool. When omitting EDTA, expression of genes known as signature genes for iron deficiency, FIT1, bHLH100, IMA1, IRT1 was strongly enhanced in lcmt1. Although an iron starvation response was induced, Fe homeostasis was apparently maintained by slowed growth in lcmt1 and the Fe level related to tissue dry weight was not changed. Among genes induced in lcmt1 were also the Zn induced gene ZIF1, and heat shock protein HSP90-1. Concentrations of non-iron transition metals, Cu, Mn and Zn, increased significantly in response to lack of EDTA for both lcmt1 and WT tissue, and especially the growth of lcmt1 was strongly hampered. Conclusions: Presence of the LCMT1 gene was necessary to cope efficiently with an imbalance in the micronutrients, heat stress, and oxidative stress. Methylation of PP2A appears important to ameliorate the toxic effects of metals present in unfavourable high concentrations as well as heat or oxidative stress. The experiments establish LCMT1 as a key component in broad stress tolerance.

Download Full-text

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury

Sensors ◽

10.3390/s20030598 ◽

2020 ◽

Vol 20 (3) ◽

pp. 598 ◽

Cited By ~ 1

Author(s):

Marialuisa Siepi ◽

Rosario Oliva ◽

Filomena Battista ◽

Luigi Petraccone ◽

Pompea Del Vecchio ◽

...

Keyword(s):

Tryptophan Residue ◽

Histidine Residues ◽

Turn On ◽

Dansyl Group ◽

C Terminus ◽

Molecular Behavior ◽

Fluorescent Peptide ◽

Acetyl Group ◽

High Concentrations ◽

Fluorescent Moiety

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

Download Full-text

Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides

Blood ◽

10.1182/blood.v74.2.743.743 ◽

1989 ◽

Vol 74 (2) ◽

pp. 743-750 ◽

Cited By ~ 17

Author(s):

PJ Haidaris ◽

CW Francis ◽

LA Sporn ◽

DS Arvan ◽

FA Collichio ◽

...

Keyword(s):

Rna Processing ◽

Plasma Fibrinogen ◽

Chain Gene ◽

Post Translational Modification ◽

Gamma Chain ◽

Specific Expression ◽

Human Fibrinogen ◽

Alternative Processing ◽

C Terminus ◽

Processing Event

Abstract The gamma chain of human fibrinogen is heterogeneous in length at the C- terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408–416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337–411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408–416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma- chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3′ RNA processing events.

Download Full-text

Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus

Biochemical Journal ◽

10.1042/bj2660083 ◽

1990 ◽

Vol 266 (1) ◽

pp. 83-90 ◽

Cited By ~ 12

Author(s):

A T Merryweather ◽

J M Crampton ◽

H Townson

Keyword(s):

Culex Quinquefasciatus ◽

Resistant Strain ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Starch Gel Electrophoresis ◽

Post Translational Modification ◽

Highly Active ◽

High Concentrations ◽

Starch Gels

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

Download Full-text

Isolation and patial sequence of bovine cDNA clones for the high-moility-group protein (HMG-1)

Bioscience Reports ◽

10.1007/bf01120823 ◽

1984 ◽

Vol 4 (1) ◽

pp. 49-57 ◽

Cited By ~ 19

Author(s):

Brian Pentecost ◽

Gordon H. Dixon

Keyword(s):

New York ◽

Dna Sequences ◽

Complete Sequence ◽

Cdna Clones ◽

Academic Press ◽

Coding Region ◽

Chromosomal Proteins ◽

C Terminus ◽

Group Protein

Several cloned ds cDNAs containing bovine HMG-1 sequences have been isolated from a ds cDNA library prepared from the poly(A)+ mRNA fraction of bovine testis using a pool of synthetic 17-rneric oligo-deoxyribonucleotides with the sequence α2 selected to be complementary to a region of the coding sequence corresponding to the relatively unambiguous amino acid sequence, Glu-Met-Trp-Asn-Asn-Thr. Determination of the DNA sequences in these clones indicates that they represent the 3′ half of the HMG-1 message and contain an unusually long putative 3′ untranslated region of 480 nucleotides. The sequence of the coding region corresponding to the 99 amino acids at the C-terminus of HMG-I has been determined and largely confirms the published primary sequence in this region (Walker 3M, (1982) in: The HMG Chromosomal Proteins, Academic Press, London & New York, pp. 69–88). In addition the cDNA sequence provides a complete sequence of the 30 residue polyacidic region and shows that the nucleotide sequence in this region is a repeating one and that the polyacidic domain comprises the C-terminus of the protein.

Download Full-text

Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Nucleic Acids Research ◽

10.1093/nar/gkt1171 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2736-2749 ◽

Cited By ~ 49

Author(s):

Kirsten E. Robinson ◽

Jillian Orans ◽

Alexander R. Kovach ◽

Todd M. Link ◽

Richard G. Brennan

Keyword(s):

Fluorescence Quenching ◽

Rna Binding ◽

Tryptophan Fluorescence ◽

Binding Motif ◽

Gram Positive ◽

C Terminus ◽

Specific Regulation ◽

Rna Mapping ◽

Rna Interaction ◽

Tryptophan Fluorescence Quenching

Abstract Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

Download Full-text