Isolation and Sequencing of Human Lamin β cDNA: Mammalian Lamins A, B and C Contain Leucine Heptad Repeats

ABSTRACT Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

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p230 is associated with vesicles budding from the trans-Golgi network

Journal of Cell Science ◽

10.1242/jcs.109.12.2811 ◽

1996 ◽

Vol 109 (12) ◽

pp. 2811-2821 ◽

Cited By ~ 7

Author(s):

P.A. Gleeson ◽

T.J. Anderson ◽

J.L. Stow ◽

G. Griffiths ◽

B.H. Toh ◽

...

Keyword(s):

Brefeldin A ◽

Immunogold Labeling ◽

Coated Vesicles ◽

Specific Marker ◽

Very High Frequency ◽

Trans Golgi Network ◽

Golgi Membranes ◽

Vesicle Biogenesis ◽

Heptad Repeats ◽

Golgi Network

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

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Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics

Glycobiology ◽

10.1093/glycob/cwaa052 ◽

2020 ◽

Cited By ~ 5

Author(s):

Dapeng Zhou ◽

Xiaoxu Tian ◽

Ruibing Qi ◽

Chao Peng ◽

Wen Zhang

Keyword(s):

Mass Spectrometry ◽

Insect Cells ◽

Mass Spectrometry Analysis ◽

Native Form ◽

Spectrometry Analysis ◽

Peptide Epitopes ◽

Central Helix ◽

The Us ◽

Accessible Surface ◽

Heptad Repeats

Abstract Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope and sugar modifications close to the peptide either strengthen or do not hinder the binding.

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Reductionist Approach in Peptide-Based Nanotechnology

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-062917-012541 ◽

2018 ◽

Vol 87 (1) ◽

pp. 533-553 ◽

Cited By ~ 19

Author(s):

Ehud Gazit

Keyword(s):

Self Assembly ◽

Electrical Characteristics ◽

Binding Agents ◽

Dna Binding Agents ◽

Ordered Nanostructures ◽

Principal Directions ◽

Heptad Repeats ◽

Β Sheet ◽

Reductionist Approach ◽

Proteins And Peptides

The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

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JMJD5 couples with CDK9 to release the paused RNA polymerase II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005745117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19888-19895

Author(s):

Haolin Liu ◽

Srinivas Ramachandran ◽

Nova Fong ◽

Tzu Phang ◽

Schuyler Lee ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Pol Ii ◽

Transcription Start Sites ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Pol Ii Pausing ◽

Carboxyl Terminal ◽

Heptad Repeats

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

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Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

The EMBO Journal ◽

10.1002/j.1460-2075.1993.tb05992.x ◽

1993 ◽

Vol 12 (8) ◽

pp. 3227-3236 ◽

Cited By ~ 57

Author(s):

S. Alberti ◽

S. Oehler ◽

B. von Wilcken-Bergmann ◽

B. Müller-Hill

Keyword(s):

Genetic Analysis ◽

Lac Repressor ◽

Helical Bundle ◽

Heptad Repeats

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Intramolecular Repression of Mouse Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.906 ◽

1998 ◽

Vol 18 (2) ◽

pp. 906-918 ◽

Cited By ~ 56

Author(s):

Thomas Farkas ◽

Yulia A. Kutskova ◽

Vincenzo Zimarino

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Coiled Coil ◽

Heat Shock Factor ◽

Heat Shock Factor 1 ◽

Reticulocyte Lysate ◽

Heptad Repeats ◽

Intramolecular Mechanism

ABSTRACT The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.

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