GABAA Receptor Trafficking-Mediated Plasticity of Inhibitory Synapses

Proper developmental, neural cell-type-specific, and activity-dependent regulation of GABAergic transmission is essential for virtually all aspects of CNS function. The number of GABAA receptors in the postsynaptic membrane directly controls the efficacy of GABAergic synaptic transmission. Thus, regulated trafficking of GABAA receptors is essential for understanding brain function in both health and disease. Here we summarize recent progress in the understanding of mechanisms that allow dynamic adaptation of cell surface expression and postsynaptic accumulation and function of GABAA receptors. This includes activity-dependent and cell-type-specific changes in subunit gene expression, assembly of subunits into receptors, as well as exocytosis, endocytic recycling, diffusion dynamics, and degradation of GABAA receptors. In particular, we focus on the roles of receptor-interacting proteins, scaffold proteins, synaptic adhesion proteins, and enzymes that regulate the trafficking and function of receptors and associated proteins. In addition, we review neuropeptide signaling pathways that affect neural excitability through changes in GABAAR trafficking

The Mechanism of Budding of Retroviruses from Cell Membranes

Retroviruses have evolved a mechanism for the release of particles from the cell membrane that appropriates cellular protein complexes, referred to as ESCRT-I, -II, -III, normally involved in the biogenesis of multivesicular bodies. Three different classes of late assembly (L) domains encoded in Gag, with core sequences of PPXY, PTAP, and YPXL, recruit different components of the ESCRT machinery to form a budding complex for virus release. Here, we highlight recent progress in identifying the role of different ESCRT complexes in facilitating budding, ubiquitination, and membrane targeting of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus, type 1 (HIV-1). These findings show that retroviruses may adopt parallel budding pathways by recruiting different host factors from common cellular machinery for particle release

Rescue of HIV-1 Release by Targeting Widely Divergent NEDD4-Type Ubiquitin Ligases and Isolated Catalytic HECT Domains to Gag

Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release

HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding

Investigating the Roles of NEDD4.2s and Nef in the Release and Replication of HIV-1: A Dissertation

Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature

Signals involved in protein intracellular sorting

“…Confusion appears to occur just after the articulation of a major conceptual advance that served to greatly clarify a problem of exceptional importance.”- Ira Mellman, 1996. What could be more fitting than the domain of protein trafficking to elucidate the above statement made by one of the several pioneers in the field? Ever since the pioneering groundwork laid down by Blobel and colleagues, emphasising protein translocation across intracellular membranes, the field of protein trafficking has been a playground of debates, dogma-reversals and rediscoveries. The possession of a valid cellular address tag is the basic requirement for the delivery of a given protein at its intracellular destination. However, the complexity involved in the foray of proteins from their site of synthesis to their site of function is within the scope of no comprehensive treatise. In this thesis, the work done on two individual transport steps of two different proteins has been summarised. In the first part of this thesis, the trafficking of the cation-dependent (CD-) mannose 6- phosphate receptor (MPR) has been studied. The CD-MPR cycles between the TGN and the plasma membrane, through the early and late endosomal compartments. It performs the important function of transport of lysosomal enzymes to lysosomes, a process which ensures the correct biogenesis of lysosomes. However, it is important that the receptor itself be excluded from lysosomes and safely retrieved to the TGN from late endosomes in order to avoid degradation in lysosomes. This is essential to ensure that the CD-MPR is available to support several rounds of lysosomal enzyme transport. This retrieval step has been shown to depend on a pair of aromatic residues F18W19 in the cytoplasmic tail of the receptor. Mutation of the residues to alanines has been shown to result in massive mislocalisation of the CD-MPR in lysosomes, the W19 residue being more crucial to this function and the F18 residue playing a contributory role. The retrieval has also been shown to take place in a Rab9 dependent manner using the cytosolic adaptor protein TIP47 (Tail Interacting Protein of 47 kDa). TIP47 specifically interacts with the diaromatic motif to effect this transport step. In this study, we demonstrated a strict requirement for di-aromaticity at the positions 18 and 19 of the cytosolic tail of the CDMPR both for correct intracellular sorting in vivo and optimal TIP47 interaction in vitro, thus demonstrating the significance of the di-aromatic motif in endosomal sorting and establishing the highly specific nature of this interaction. This also established a paradigm for the CD-MPR as a representative member of a generic family of diaromatic motif containing proteins. The second part of this thesis deals with the trafficking of the human mannose 6- phosphate uncovering enzyme (UCE). The recognition of the mannose 6-phosphate tag on lysosomal enzymes by the MPRs is facilitated by UCE which exposes the recognition signal on the lysosomal enzymes in a two-step enzymatic reaction: the first starts in the cis-Golgi and is mediated by a phosphotransferase and the second, mediated by UCE, occurs in the TGN. At steady state, UCE is mostly localised to the TGN and it cycles between the TGN and the plasma membrane. It is rapidly internalised from the surface in a clathrin dependent endocytic pathway and the internalisation has been shown to be mediated by a critical tyrosine-488 residue in its cytoplasmic tail. The transmembrane domain and first 11 residues of the cytoplasmic tail of UCE have been shown to be involved in its TGN retention. In this study, we identified the residues involved in TGN exit of UCE using a combination of biochemical and confocal immunofluorescence methods. Using a high dimensional neural network capable of identifying differences between images not visible to the eye, we determined that the residues 492QEMN were involved in TGN exit of UCE. The same method was also used to analyse the individual contribution of each amino acid in the sequence and it was found that residue Q492 is the most important to the exit function while residues M494 and N495 also contribute. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in lysosomal biogenesis. With these two analyses, we contributed to a better understanding of signal sequences involved in intracellular protein trafficking of two related proteins both involved in lysosomal biogenesis

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the “late domain”) binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs; residues 222–777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222–777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding