Structural and Functional Study of Apoptosis-linked Gene-2·Heme-binding Protein 2 Interactions in HIV-1 Production

Abstract

Edited by Dennis VoelkerIn the HIV-1 replication cycle, the endosomal sorting complex required for transport (ESCRT) machinery promotes viral budding and release in the late stages. In this process, the ESCRT proteins, ALIX and TSG101, are recruited through interactions with HIV-1 Gag p6. ALG-2, also known as PDCD6, interacts with both ALIX and TSG101 and bridges ESCRT-III and ESCRT-I. In this study, we show that ALG-2 affects HIV-1 production negatively at both the exogenous and endogenous levels. Through a yeast two-hybrid screen, we identified HEBP2 as the binding partner of ALG-2, and we solved the crystal structure of the ALG-2⅐HEBP2 complex. The function of ALG-2⅐HEBP2 complex in HIV-1 replication was further explored. ALG-2 inhibits HIV-1 production by affecting Gag expression and distribution, and HEBP2 might aid this process by tethering ALG-2 in the cytoplasm.The endosomal sorting complex required for transport (ESCRT) 3 machinery functions in HIV-1 budding and release in the late stages of viral replication (1-3). In this process, ALIX (ALG-2 interacting protein X) and TSG101 (tumor susceptibility gene 101), components of ESCRT-III and ESCRT-I, are recruited to the budding site of HIV-1 through interactions with HIV-1 Gag p6 (4 -8). This promotes the release of virion particles (9) with the help of AAA-ATPase VPS4B (vacuolar protein sorting 4 homolog B) (10, 11). The penta-EF-hand protein ALG-2 (apoptosis-linked gene-2, also known as PDCD6) participates in the process by interacting with both ALIX (12, 13) and TSG101 (14) in a calcium-dependent manner and bridges ESCRT-III and ESCRT-I through dimerization (15)(16)(17). However, the function of ALG-2 in viral replication, especially that of HIV-1, remains unknown.ALG-2 was first identified in a functional screen for anti-apoptotic proteins involved in T-cell receptor, Fas, and glucocorticoid-induced cell death (18). It was defined as a proapoptotic molecule because expression of its antisense strand led to rescue from cell death (19). However, ALG-2 knock-out mice are viable and exhibit normal T-cell development (20), which suggested that other redundant proteins might exist in mammalian cells. ALG-2 is a penta-EF-hand protein (13, 15), containing five repeats of the EF-hand motif, and coordinates three calcium ions via EF1, EF3, and EF5. However, the coordination of calcium by EF5 is weak and may be physiologically unimportant. ALG-2 exists as a dimer, which might be critical for its function as an adaptor molecule in signal transduction (15). ALG-2 can interact with dozens of proteins through several pockets (pockets 1-3) on its molecular surface. The binding motif for ALG-2, designated as the ALG-2-binding site (ABS), is a short sequence rich in Pro, Gly, and Tyr. Two typical ABS sequences have been described. ABS-1, which is represented as PPYPXXPGYP (X represents any residue) and occupies pocket 1 on the ALG-2 surface, is found in ALIX and PLSCR3 (6), whereas ABS-2, containing a PXPGF sequence that binds pocket 3, is found in SEC31 and PLSCR3 (1...

Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes

“…Moreover, Tsg101 and Alix were reported to be necessary for the formation of viruses and were associated with viral proteins such as Gag p6. 26) We speculate that Tsg101 and Alix in exosomes may interact with various components in lipid raft domains or core structures which allows them to be resistant to NP-40 or Triton X-100. Several other studies employing detergents reported on the stability of exosomes.…”

Deregulated ALG‐2/HEBP2 axis alters microtubule dynamics and mitotic spindle behavior to stimulate cancer development

“…4′,6‐diamidino‐2‐phenylindole (DAPI) were from Sigma–Aldrich. Human ALG‐2 siRNAs and plasmids expressing various tagged ALG‐2 and HEBP2 were described previously (Ma et al, ; Yamasaki et al, ), and their mutants were constructed by using the site‐directed mutagenesis kit (TransGen, Beijing, China).…”

“…The expression of HEBP2 has been reported to correlate with the efficacy of neoadjuvant radiochemotherapy and predict outcomes in patients with locally advanced squamous cell esophageal cancer (Farkas et al, ). Recently, the crystal structure of the ALG‐2/HEBP2 complex has been solved, which suggests a function for this complex in human immunodeficiency virus replication (Ma et al, ). However, whether and how the ALG‐2/HEBP2 complex is involved in cancer development are unknown.…”

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“…Specifically, exosomes originate from endosomal intralumenal vesicles and are enriched in tetraspanins including CD9, CD63, and CD81 . Many other proteins are associated with the ESCRT complex, such as HSC70, TSG101, Alix, and Flotillin‐1, reported to mark subtypes of EVs . However, a new study using human DCs to characterize markers per EV subtype discovered that some conventionally accepted exosome markers including MHC I or II, flotillin, and HSC70, are present in multiple EV subpopulations .…”

“…44 Many other proteins are associated with the ESCRT complex, such as HSC70, TSG101, Alix, and Flotillin-1, reported to mark subtypes of EVs. 110,111 However, a new study using human DCs to characterize markers per EV subtype discovered that some conventionally accepted exosome markers including MHC I or II, flotillin, and HSC70, are present in multiple EV subpopulations. 17 Analyses of EVs isolated by CD9-, CD63-, or CD81-specific antibodies by qualitative and quantitative proteomic comparison suggested that a disintegrin and metalloprotease 10 (ADAM10), EH domain containing 4 (EHD4), syntenin-1, and TSG101 only reside in small EVs (sEVs), with syntenin-1 and TSG101 being specific for the tetraspanin-enriched sEVs and representing "bona fide markers" for exosomes.…”