Cell Biology

The ubiquitin-conjugating enzyme UBE2D/eff maintains a youthful proteome and ensures protein quality control during aging

Liam C Hunt
Kudzai Nyamkondiwa
Anna Stephan
Jianqin Jiao
Kanisha Kavdia
Vishwajeeth Pagala
Junmin Peng
Fabio Demontis author has email address

Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA

https://doi.org/10.7554/eLife.94739.2

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Figures and data

RNAi for E2 ubiquitin-conjugating enzymes and associated E3s and DUBs modulates pathogenic huntingtin-polyQ aggregates in the Drosophila retina
(A) GFP-tagged huntingtin-polyQ (Htt-polyQ72-GFP) driven with GMR-Gal4 leads to GFP-fluorescent Htt protein aggregates in the retina at 30 days of age. Compared to negative controls (mCherry^RNAi and luciferase^RNAi), RNAi for E2 ubiquitin-conjugating enzymes, associated deubiquitinating enzymes (DUBs), and E3 ubiquitin ligases (E3s) modulates the amount of Htt-polyQ72-GFP aggregates. Specifically, RNAi for Ubc6 (homologous of UBE2A/B) and for its associated E3 ubiquitin ligases Ubr4 and Kcmf1 increases the amount of Htt-polyQ72-GFP protein aggregates. Similar increases are also seen with knockdown of eff/UBE2D and the associated E3 HUWE1, and with RNAi for Ubc10/UBE2L3, Ubc84D/UBE2L6, and for the associated E3 enzyme BTBD8. Positive controls include Akt RNAi, which reduces protein aggregates, and Hsp83 RNAi, which increases them. (B-C) Quantitation of the total area of Htt-polyQ72-GFP aggregates modulated by RNAi for E2s (B) and for associated E3s and DUBs (C). Relative fold changes compared to control RNAi interventions are shown; n=5 (biological replicates), SD. Each data point in the graph represents a single RNAi targeting the corresponding gene. For each RNAi, the mean of 5 biological replicates is shown, with each biological replicate representing a single eye (each from a distinct animal).
(D) qRT-PCR indicates that there are no changes in GFP and Htt mRNA levels upon eff knockdown compared to control RNAi, indicating that eff RNAi does not modulate the amount of GFP-tagged huntingtin-polyQ aggregates via changes in the expression of Htt-polyQ72-GFP transgenes; n=3 (biological replicates) and SEM.
(E) Levels of Htt-polyQ72-GFP aggregates detected by western blot with anti-GFP antibodies identify Htt-polyQ72-GFP monomers (∼50 kDa) and high-molecular-weight (HMW) assemblies of Htt-polyQ72-GFP in the stacking gel (>250 kDa). Knockdown of eff/UBE2D increases the levels of HMW Htt-polyQ72-GFP; n=3 (biological replicates), SEM, and p-values (one-way ANOVA) are indicated, with **p<0.01, compared to mcherry^RNAi.

Retinal degeneration induced by knockdown of Ubc6 and eff is rescued by expression of the respective human homologs UBE2B and UBE2D2/4
(A) Compared to control RNAi, knockdown of Ubc6 and eff causes retinal degeneration, as indicated by the “rough eye” phenotype (R), by black necrotic patches, and/or by areas with depigmentation. Human UBE2B rescues degeneration induced by RNAi for its Drosophila homolog Ubc6 but not induced by RNAi for the unrelated E2 enzyme eff. Likewise, human UBE2D2 and UBE2D4 rescue retinal degeneration induced by knockdown of their Drosophila homolog eff. (B) Htt-polyQ72-GFP aggregates increase in Drosophila retinas with eff RNAi and this is rescued by human UBE2D2 but not by mcherry; n=3 (biological replicates), SEM, and p-values (one-way ANOVA) are indicated, with ***p<0.001. (C) Similarly, high-molecular-weight assemblies of Htt-polyQ72-GFP detected in the stacking gel increase with eff RNAi compared to control RNAi but this is rescued by hUBE2D2 versus control mcherry expression.

RNAi for the E2 enzyme eff/UBE2D impairs muscle protein quality control during aging
(A) TMT mass spectrometry of Drosophila skeletal muscle from old versus young w¹¹¹⁸ flies at 8 vs. 1 week old; n=5 biological replicates. The x axis reports the Log2FC whereas the y axis reports the –log10(p-value). The E1 enzyme UBA1 and the E2 enzymes that are detected are indicated, including UBE2D/eff; n=5 (biological replicates).
(B) The protein levels of UBE2D/eff significantly decline with aging (8 versus 1 week-old) in skeletal muscle; n=5 (biological replicates), SD, and p-values (Student’s t-test).
(C-D) Immunostaining of Drosophila skeletal muscle at 3 weeks of age indicates that eff/UBE2D RNAi impairs protein quality control, as indicated by the higher age-related accumulation of aggregates of poly-ubiquitinated proteins, compared to control RNAi. The scale bar is 20 μm. In (C), n=10 (biological replicates), SD, and p-values (Student’s t-test).
(E-F) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that eff RNAi impedes proteostasis, as indicated by higher levels of detergent-soluble and insoluble poly-ubiquitinated proteins compared to control RNAi (E). A similar increase is also found for the detergent-soluble levels of Ref(2)P/p62 (E). n=3 (biological replicates), SEM, **p<0.01, ns=not significant (one-way ANOVA).

Defects in proteostasis and lifespan caused by muscle-specific eff/UBE2D RNAi are partially rescued by the expression of human UBE2D2
(A-B) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that defects in proteostasis due to eff RNAi can be partially rescued by its human homolog UBE2D2, as indicated by the normalization of the detergent-soluble levels of poly-ubiquitinated proteins and Ref(2)P/p62. However, hUBE2D2 does not impact their detergent-insoluble levels, indicating that hUBE2D only partially rescues defects induced by eff/UBE2D RNAi. In (B), n=3 (biological replicates), SEM, **p<0.01 (one-way ANOVA). (C) Muscle-specific eff/UBE2D RNAi reduces eff mRNA levels; n=3 (biological replicates), SEM, **p<0.01 (one-way ANOVA). (D) Two distinct RNAi lines targeting eff/UBE2D reduce lifespan compared to control RNAi (p<0.001, log-rank test). (E) The decline in organismal survival due to eff/UBE2D RNAi in muscle is partially rescued by transgenic hUBE2D expression compared to control mcherry overexpression (p<0.001, log-rank test).

Deep-coverage TMT mass spectrometry identifies the proteins modulated by UBE2D/eff knockdown in Drosophila skeletal muscle
(A-B) TMT mass spectrometry of Drosophila skeletal muscle with eff/UBE2D knockdown versus control RNAi (A) and overexpression of human UBE2D2 versus control mcherry (B). The x axis reports the Log2FC whereas the y axis reports the –log10(p-value). Examples of regulated proteins are shown in red. (C) Cross-comparison of the Log2FC induced by eff RNAi versus control RNAi (x axis) with the Log2FC of hUBE2D2 versus control mcherry (y axis). (D) Cross-comparison of the Log2FC induced by eff RNAi + mcherry versus control (x axis) with the Log2FC of eff RNAi + hUBE2D2 versus control (y axis) indicates that some of the protein changes induced by eff RNAi are rescued by hUBE2D2. (E) GO term analysis of protein categories that are enriched among upregulated and downregulated proteins in response to eff/UBE2D2 RNAi. The enrichment scores and number of regulated DAVID IDs are shown. (F-G) Examples of regulated proteins modulated by eff RNAi and hUBE2D-mediated rescue include protein changes that may drive derangement of proteostasis, such as Arc1/2 upregulation (F) as well as protein changes that are protective and likely compensatory, such as proteasome components and chaperones (G); n=3 (biological replicates), SEM, *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA).

Knockdown of the ubiquitin-conjugating enzyme UBE2D/eff drives proteomic changes associated with aging in Drosophila skeletal muscle
(A-D) Cross-comparison of TMT mass spectrometry data from Drosophila skeletal muscle identifies substantial overlap in the proteomic changes induced by UBE2D/eff knockdown in young age compared to the changes that are induced by aging. The x axis displays the significant (p<0.05) changes (log₂FC) induced in skeletal muscle by eff RNAi (Mhc>eff^RNAi) compared to control mcherry RNAi (Mhc>mcherry^RNAi) at 2 weeks of age (n=3 biological replicates/group). The y axis reports the significant (p<0.05) changes induced by aging in the skeletal muscle of control flies (w¹¹¹⁸) when comparing 8 weeks (old) versus 1 week (young), with n=5 biological replicates/group. Among the proteins that are significantly regulated (p<0.05) by both UBE2D/eff knockdown and aging, ∼70% are consistently regulated, i.e. either upregulated (B, 51%) or downregulated (A, 18.5%) by both, whereas the remaining ∼30% is regulated oppositely by UBE2D/eff RNAi versus aging (A, D). The protein categories that are over-represented in each group are indicated in (A-D) alongside the enrichment score. Representative proteins that are significantly regulated by UBE2D/eff RNAi in a consistent or discordant manner are shown in the graph and include Arc1 and Arc2. (E) Venn diagrams representing the overlap in the regulation of protein levels by aging and eff^RNAi. These graphs were obtained from the list of significantly regulated proteins modulated by aging and eff^RNAi (Supplementary Table S4). The threshold of log₂FC>0.3 and <-0.3 was further applied for selecting up- and down-regulated proteins.

The E2 enzyme UBE2D/eff is necessary to preserve proteostasis and to maintain a youthful proteome composition in skeletal muscle during aging
The ubiquitin-conjugating enzyme UBE2D/eff has a key role in proteostasis in skeletal muscle: UBE2D/eff levels decline during aging and reproducing such UBE2D/eff knockdown from a young age causes a loss in protein quality control and a consequent precocious surge in the levels of insoluble poly-ubiquitinated proteins, which normally accumulate only in old age because of age-associated defects in proteostasis. Proteomics surveys indicate that UBE2D/eff knockdown rewires the proteome similar to aging, and that the UBE2D/eff^RNAi-induced changes are rescued by transgenic expression of its human homolog UBE2D2. Altogether, these findings indicate that UBE2D/eff is necessary to maintain a youthful proteome and to ensure muscle protein quality control during aging.

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