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. 2009 Oct;1792(10):1027-35.
doi: 10.1016/j.bbadis.2009.08.009. Epub 2009 Aug 22.

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Jian Jiao  1 , Kaihua Sun, Will P Walker, Pooneh Bagher, Christina D Cota, Teresa M Gunn
Affiliations

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Jian Jiao et al. Biochim Biophys Acta. 2009 Oct.

Abstract

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a "PSAP" motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.

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Figures

Fig. 1
Fig. 1
MGRN1 associates with TSG101. (A) Schematic of TSG101 and MGRN1 proteins. Regions of each protein found to interact with the other in yeast two-hybrid assays are indicated by dashed lines. UEV: ubiquitin E2 variant domain; PRD: proline rich domain; CC: coiled-coil domain; SB: steadiness box domain; RF: C3HC4 ring finger domain. The position of late-viral P[S/T]AP domains in TSG101 and MGRN1 are also indicated. (B) MGRN1 associates with TSG101. IP of lysates from untransfected HEK293T cells for TSG101 (left panel) or MGRN1 (right panel) followed by IB for MGRN1 (left panel) or TSG101 (right panel) confirmed association of the endogenous proteins. Clean-blot detection reagent was used in lieu of secondary antibody for TSG101 IB of MGRN1 IP products to eliminate the heavy chain IgG band that otherwise obscures the TSG101 signal.
Fig. 2
Fig. 2
A conserved PSAP motif in MGRN1 mediates its association with TSG101. (A) Vertebrate MGRN1 homologs contain a PSAP “late-viral” motif that is not found in the related protein, MGRN2 (RNF157) or invertebrate MGRN homologs. (B) HEK293T cells were transfected with Myc-tagged TSG101 and control GFP, MGRN2-GFP, wild-type MGRN1-GFP, or mutant MGRN1(ASAA)-GFP (PSAP motif mutated to ASAA). Lysates were subjected to IP using an antibody against Myc (to IP TSG101) and blotted for GFP. Only wild-type MGRN1-GFP coimmunoprecipitated with TSG101, indicating that the PSAP motif is required for their association. (C) Schematic showing protein structures of and amino acid identity between MGRN1 and MGRN2. MGRN2 does not contain a PSAP motif.
Fig. 3
Fig. 3
MGRN1 multimonoubiquitinates TSG101. (A) MGRN1 ubiquitinates TSG101. Duplicate sets of HEK293T cells were transfected with indicated plasmids. One set of plates was treated with proteasome inhibitor MG132 prior to collection of lysates. Lysates were subjected to IP for endogenous TSG101 and blotted for HA (ubiquitin). Regardless of MG132 treatment, TSG101 was multiubiquitinated in cells expressing empty GFP vector or wild-type MGRN1-GFP, with a stronger signal in cells overexpressing MGRN1. Very little ubiquitinated TSG101 was detected in cells expressing catalytically inactive MGRN1 (MGRN1(AVVA)-GFP) or siRNA against Mgrn1. (B) Dose-dependent effect of MGRN1 on TSG101 ubiquitination. HEK293T cells were transfected with indicated amounts (μg) of each plasmid. Lysates were blotted for Myc (TSG101), GFP (GFP or tagged MGRN1) and GAPDH (control for protein loading). A smear of ubiquitinated TSG101 was detected in cells expressing MGRN1, with a stronger signal in cells expressing higher amounts of MGRN1. The intensity of the signal for multiubiquitinated TSG101 (Myc-TSG101-(Ub)n) was much reduced in cells expressing catalytically inactive MGRN1 (AVVA mutant). (C) MGRN1 multimonoubiquitinates TSG101. HEK293T cells were transfected with indicated plasmids and lysates subjected to IP for endogenous TSG101. IP products were blotted for GFP to detect ubiquitin-K0 (contains no lysine residues to allow for the formation of polyubiquitin chains). A significant increase in multimonoubiquitinated TSG101 was observed in cells overexpressing MGRN1. Lysates were blotted for HA (to detect HA-MGRN1; HA produced from empty vector ran off the bottom of the gel) and TSG101 to confirm expression. (D) HEK293T cells were transfected with indicated plasmids. Lysates were subjected to IP for GFP (MGRN1) and blotted for Myc (TSG101) to demonstrate that catalytically inactive MGRN1 (AVVA mutant) not only still binds TSG101, but binds it more tightly than wild-type MGRN1: Myc-TSG101 expression was equal in the lysates prior to IP but much stronger in the IP lane from cells expressing MGRN1(AVVA)-GFP.
Fig. 4
Fig. 4
Age- and MGRN1-dependent changes in TSG101 ubiquitination in the mouse brain. (A) Immunoblotting (IB) of whole brain protein lysates from wild-type (wt), Mgrn1 heterozygous (het) and Mgrn1 null mutant mice for TSG101. In wild-type mice, high levels of multiubiquitinated TSG101 (TSG101-(Ub)n) were observed in lysates from 1-month-old animals, but most TSG101 expression in older (3-month-old) animals was unmodified or monoubiquitinated. At 1-month of age, the levels of multiubiquitinated TSG101 were lower in Mgrn1 null mutant brains than in wild-type brains, but at 3-months of age, there was more multiubiquitinated TSG101 in the brains of mutant mice. At both ages, heterozygotes had levels intermediate to those of null mutants and wild-type mice. A 3-min exposure is shown for 1 month samples, a 1-min exposure for 3-month samples (3-min exposure of both shown in Supplemental Fig. S3B). Multiubiquitinated TSG101 was observed in samples from 3-month-old mice when blots were imaged longer (not shown). (B) Brain lysates from 6-month-old wild-type (wt) and Mgrn1 null mutant mice were subjected to IP for TSG101. Duplicate blots were immunoblotted for TSG101 and ubiquitin (FK2 antibody, which recognizes mono- and poly-ubiquitinated proteins), respectively. Both antibodies detected the same high molecular weight bands, indicating that they represent multiubiquitinated TSG101.
Fig. 5
Fig. 5
EGFR, which requires functional TSG101 for its endosomal transport to the lysosome to be degraded, accumulates in the brains of Mgrn1 null mutant mice. Brain protein lysates from wild-type (wt) and Mgrn1 null mutant animals of the indicated ages were subjected to IB for EGFR. Protein loading is indicated by IB for GAPDH.
Fig. 6
Fig. 6
Loss of MGRN1 results in the accumulation of insoluble, high molecular weight (multiubiquitinated) forms of TSG101. Sequential extraction of brain proteins from 1- and 6-month-old wild-type (wt) and Mgrn1 null mutant mice was performed to isolate proteins based on their solubility (proteins in Triton X-100 fractions are more soluble than those in SDS fractions). Fractions were subjected to IB to detect unmodified and ubiquitinated TSG101 (TSG101-(Ub)n). The same blots were imaged for 1 min (top panels) and 10 min (bottom panels), and blotted for GAPDH as a loading control. In the brains of young animals, multiubiquitinated TSG101 was predominantly observed in the SDS fraction. In older animals, multiubiquitinated TSG101 was observed in both fractions, although more was present in the SDS fraction in the brains of Mgrn1 null mutants than in wild-type mice.
Fig. 7
Fig. 7
Overexpression of neither wild-type nor catalytically inactive MGRN1 significantly affects HIV-1 GAG release from cells. HEK293T cells were cotransfected with an HIV-1 GAG p55 expression construct and either a GFP control, a positive control for budding inhibition (GFP-VPS4(EQ)), or GFP fusion constructs expressing wild-type MGRN1 or catalytically inactive MGRN1 (AVVA mutant). (A) Representative immunoblots from 1 of 3 replicate experiments. Top panel: GAG IB of virus-like particle (VLP) preps from media of transfected cells. Middle panel: GAG IB of cell lysates of transfected cells. Bottom panel: non-specific bands from upper region of cell lysate blot demonstrate even loading. (B) Graphical representation of the effect of each treatment on VLP budding, averaged from 3 replicate experiments. Relative budding index (y axis) represents the ratio of GAG signal in VLPs to GAG signal in cell lysates of each transfection relative to that of GAG + GFP control samples. VPS4(EQ) reduced GAG release (p < 0.02) while neither wild-type nor catalytically inactive MGRN1 had a significant effect (p > 0.3). The standard error of the GAG + GFP transfected samples is 0 because the budding index of these samples is defined as 1 within each experiment.
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