Identification of Histone Peptide Binding Specificity and Small-Molecule Ligands for the TRIM33α and TRIM33ß Bromodomains.

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1ß), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33ß, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33ß, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31-27K18Ac, and bind preferentially to H31-27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.

Protocol for generation of human peptide-specific primary CD8+ T cell lines.

Directly ex vivo, peptide-specific CD8+ T cells are present at relatively low frequency and are typically in a resting state. This protocol details the expansion of memory peptide-specific CD8+ T cells by in vitro stimulation, which can be subsequently characterized using a range of assays including tetramer staining and intracellular cytokine staining. For complete details on the use and execution of this protocol, please refer to Lineburg et al. (2021).

Probing for submandibular gland peptide-T receptors on leukocytes with biotinylated-Lys-[Gly](6)-SGP-T.

Submandibular gland peptide-T (SGP-T) is a potent anti-chemotactic agent for human neutrophils possessing anti-inflammatory properties. Biologically active analogues of SGP-T have been synthesized and a biotinylated form (KG(6)-SGP-T; Bio-KG(6)-SGP-T) was utilized to identify binding sites on isolated human neutrophils. Neutrophils incubated with Bio-KG(6)-SGP-T followed by phycoerythrin (PE)-avidin secondary reagent were fixed and visualized using histochemistry and flow cytometry. At doses of 10(-8) and 10(-9) M, Bio-KG(6)-SGP-T was shown to bind to neutrophils. The binding of Bio-KG(6)-SGP-T, at doses of 10(-8) and 10(-9) M, to neutrophils was abolished by a 100-fold excess of non-biotinylated peptide (KG(6)-SGP-T), but not by 100-fold excess of SGP-T. However, all peptides, dose-dependently reduced the binding of a CD16b antibody (LNK16 clone) to isolated human neutrophils. This discrepancy probably results from different preferred conformations for Bio-KG(6)-SGP-T, KG(6)-SGP-T and SGP-T, since exhaustive conformational searches revealed a high degree of overlap between alpha-Bio-KG(6)-SGP-T and KG(6)-SGP-T that was not seen with SGP-T.

Docking of peptide-T onto the D1 domain of the CD4 receptor.

Peptide T (pepT) is a segment of the human immunodeficiency virus (HIV) envelope protein gp120. The peptide competitively binds to the CD4 receptor of a subset of peripheral T lymphocytes and inhibits binding of gp120. Previous studies of this laboratory allowed the assessment of a bioactive form of the peptide and a pharmacophore for the peptide-receptor interaction. In the present study the proposed bioactive form of pepT and its (4-8) segment, the smallest pepT fragment shown to retain full activity, were docked onto the D1 domain of the CD4 receptor. The bioactive conformation of the peptides complements well a cleft on the surface of the CD4 receptor, shown to be the attachment site of gp120 from site directed mutagenesis experiments. These studies provide an improved description of the ligand-receptor pharmacophore.

Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis.

We previously reported that certain short gp120 V2 region peptides homologous to vasaoactive intestinal peptide (VIP), such as "peptide T," were potent inhibitors of gp120 binding, infectivity, and neurotoxicity. The present study shows that synthetic V2-region-derived peptides have potent intrinsic chemotaxis agonist activity for human monocytes and also act as antagonists of high-affinity (0.1 pM) gp120-mediated monocyte chemotaxis. Selectivity is shown in that peptide T is more potent at suppressing M-tropic than T-tropic gp120 chemotaxis. Peptide T was also able to suppress monocyte chemotaxis to MIP-1beta, a chemokine with selectivity for CCR5 chemokine receptors, while chemotaxis of the more promiscuous ligand RANTES was not inhibited, nor was chemotaxis mediated by SDF-1alpha. In order to determine if peptide T mediated its gp120 antagonistic effects via modulation of CCR5 receptors, RANTES chemotaxis was studied using a CCR5 receptor-transfected HOS cell line. In this case, RANTES chemotaxis was potently inhibited by V2-region-derived short peptides. Peptide T also partially suppressed (125)I-MIP1-beta binding to human monocytes, suggesting action at a subset of MIP1-beta receptors. The V2 region of gp120 thus contains a potent receptor binding domain and synthetic peptides derived from this region modulate CCR5 chemokine receptor chemotactic signaling caused by either gp120 or chemokine ligands. The results have therapeutic implications and may explain recent clinical improvements, in that HIV/gp120 actions at CCR5 receptors, such as occur in the brain or early infection, would be susceptible to peptide T inhibition.

Amygdalin binds to the CD4 receptor as suggested from molecular modeling studies.

The geometrical features of the proposed bioactive conformation of peptide T assessed by computational methods in a previous study, together with available structure-activity studies on peptide T, led us to propose a pharmacophore for the CD4-peptide T interaction. Subsequent, data base searching permitted us to identify amygdalin as a peptide T peptidomimetic.

Investigation into the intestinal metabolism of [D-Ala1] peptide T amide: implication for oral drug delivery.

The anti-AIDS drug, [D-Ala1] Peptide T amide (D-ASTTTNYT.NH2) is an octapeptide which competitively inhibits the attachment of HIV to the receptor CD4 molecule on the T-lymphocyte. The objective of the study is to investigate the degradative process of this peptide and its effective enzyme inhibitors. The metabolites of [D-Ala1] Peptide T amide in rabbit brush-border membrane vesicles at pH 6.5 are ASTT, ASTTTN, YT and Y. The sequential time-course study of each metabolite reveals that enkephalinase (EC 3.4.24.11) plays an important role in the hydrolysis of [D-Ala1] Peptide T amide to ASTT. With the addition of an enkephalinase inhibitor, thiorphan, 85% of degradation was inhibited. Aminopeptidase is also involved in its degradative process and 25% of inhibition was observed by amastatin, an aminopeptidase inhibitor. The results show that no significant difference was observed between the in situ and chronical loop perfusion studies and enzyme activities are somewhat inhibited under acidic conditions in both methods. Approx. 90% of the parent peptide remained when rats were perfused with pH 4.0 peptide solution at a flow rate of 0.123 ml/min, while only 60% was recovered when pH 6.5 peptide solution was applied. The addition of amastatin made a quadrupled increase in the amount of parent peptide recovered. A 117-fold increment was observed when thiorphan was added. The dimensionless wall permeability of this peptide was 1.19 +/- 0.16 when pH 4.0 peptide solution was used during chronical loop perfusion study. Therefore, this study suggests that [D-Ala1] Peptide T amide could be absorbed via small intestine where enzymatic degradation s a rate-limiting step for the absorption of this peptide.

Peptide T from human immunodeficiency virus does not interact with VIP receptor-effector system in immunocompetent cells of rat and mouse.

Human immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is an appreciable structural similarity between peptide T and an eight amino acid sequence of VIP, it is interesting to investigate the interaction of peptide T with the VIP receptor-effector system of immunocompetent cells from both rat and mouse. In this paper, we show the lack of interaction between peptide T and VIP receptor-effector system in peripheral blood lymphocytes, spleen lymphocytes and macrophages of rat and in macrophages of mouse. These results do not support the hypothesis that HIV through peptide T may interact with the VIP receptor-effector system present in immunocompetent cells.

Synthesis and biological activity of D-glucopyranosyl peptide T derivatives.

The solid phase procedure, based on the Fmoc (9-fluorenylmethyloxycarbonyl) chemistry, was used to prepare some peptide T analogues in which D-glucopyranosyl units are beta-O-glycosidically linked to Thr4 and/or Thr5 side chains. All glycopeptides showed significant human monocyte chemotaxis and high resistance to degradation by plasma or brain enzymes.

Verification of the interaction between peptide T and CD4 using surface plasmon resonance.

Peptide T is currently in phase II clinical trials for the treatment of AIDS-associated dementia. Its putative mode of action is inhibition of binding of the HIV envelope protein (gp120) to its cellular receptor (CD4), thus preventing viral infectivity and gp120-induced neuronal toxicity. However, a number of reports have appeared in the literature which have failed to observe any inhibitory activity of Peptide T on CD4-gp120 binding, thus casting doubt on this hypothesis. This study uses a novel biosensor technique to demonstrate that Peptide T does bind to CD4 and that this binding can be specifically inhibited by an anti-CD4 monoclonal antibody. A detailed analysis of the kinetics of the interaction is presented.

Structure-activity relationships of cyclic and linear peptide T analogues.

Using the potent cyclic peptide T analog [formula: see text] as parent compound, a series of analogues were synthesized and their potencies in a monocyte chemotaxis assay were compared with those of correspondingly modified linear peptides. Structure-activity relationships observed with cyclic compounds did not always parallel those determined with linear analogues. [formula: see text] showed the highest affinity to CD4 receptor of monocytes of any peptide thus far studied. It also proved to be highly resistant to degradation by plasma or brain enzymes.

Synthesis and binding of peptide T and aminoacyl derivatives to CD4+ lymphocytes.

1. Peptide T and four other aminoacyl derivatives of this octapeptide were synthesized on solid phase support using the Boc and Fmoc procedures. 2. The octapeptides were modified by chloroacetylation and radiolabelled by halogen exchange with 125I. 3. Purified and crude extracts of lymphocytes were used to determine the binding of the octapeptides at different concentrations.

The influence of D-Ala1-peptide T amide, an analogue of HIV glycoprotein 120 fragment, on the CD4--anti CD4 lymphocyte interaction.

D-Ala1-peptide T amide (DAPTA), synthetic analogue of the HIV glycoprotein 120 sequence, was used to study its binding to specific cellular receptor of HIV, CD4. The analogue contains eight aminoacid residues including 4 threonine residues; its molecular weight being 992. We have shown the temperature- and dose-dependent inhibitory effect of peptide T on the CD4 - anti CD4 binding. The strongest effect was noted at 37 degrees C with the peptide dose of 150 nM: 62% inhibition of binding. Other means of possible blocking of HIV attachment to the host cellular receptor are discussed.

Synthesis, metabolic stability and chemotactic activity of peptide T and its analogues.

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity relationships of peptide T-related pentapeptides.

Fifteen pentapeptide analogs of C-terminal fragment of peptide T, H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, were prepared and tested for human monocyte chemotaxis. Structure-activity studies suggest that the potent chemotactic activity of H-Thr-Thr-Asn-Tyr-Thr-OH is mediated through the polar properties of the C-terminal carboxyl group and Thr side chains at the critical positions 5 and 8, while the hydroxyl group of N-terminal Thr and its free amino function are not essential requirements for CD4 receptor interactions.