1.C.101 The HIV-1 TAT Peptide Translocator (HIV-TAT1) Family

The HIV-1 Tat protein (101 aas) contains a small region, corresponding to residues (47)YGRKKRRQRRA(57), which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner (Ciobanasu et al., 2010; Durzyńska et al. 2015). The pathway that these peptides follow for entry into the cell has been the subject of strong controversy. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. Herce and Garcia (2007) proposed a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically, the arginine residue dependency, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions (Herce and Garcia, 2007). HIV-1 Tat directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase (Lecoeur et al., 2012).

HIV-Tat is secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Zeitler et al. 2015 showed that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2, but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was required for efficient membrane pore formation by HIV-Tat oligomers. Tat can cross the membrane through an induced nanopore, the formation of which is induced by the transmembrane electrostatic potential difference (Quan et al. 2019). These properties are similar to what has been reported previously for fibroblast growth factor 2 (FGF2) (see TC# 1.A.108; Zeitler et al. 2015).

The catalytic and inhibitory effects on enzymatic digestion of Tat are associated with Ca²⁺ and Cu²⁺ ions, respectively, in response to binding interactions with trypsin. Considering the closer mimic of the real situation of HIV spread, measurements in the serum and on cells were also investigated. Transmembrane current measurements together with fluorescence microscopy imaging indicated the potential to perturb Tat transport in the serum environment and on cells (Wang et al. 2021).

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