Kawasaki I et al. (2024) Nat Commun "MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans."

Sato K, Kawasaki I, Sasaki T, Matsuda N, Sato M, Sugiura K

[Nat Commun, 2024]

In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.

Oswald, Mackenzi et al. (2020) MicroPubl Biol

Oswald, Mackenzi, Hulsey-Vincent, Heino, Dahlberg , Caroline (Lina)

[MicroPubl Biol, 2020]

Secretory and membrane-bound proteins must be properly folded and matured at the endoplasmic reticulum (ER). Despite the molecular machinery dedicated to these processes, up to 1/3 of proteins are destroyed within minutes of their synthesis (Hirsch et al. 2009; Schubert et al. 2000). Misfolded proteins in the endoplasmic reticulum can accumulate and disrupt proteostasis, which can contribute to neurodegenerative diseases. The Endoplasmic Reticulum Associated Degradation (ERAD) pathway relies on E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases to ubiquitylate misfolded proteins, signaling for degradation of these misfolded proteins by the proteasome (Vembar and Brodsky 2008). Three putative E3 ligases that are expected to be involved in ERAD in C. elegans are HRDL-1, HRD-1, and MARC-6 (Sasagawa et al. 2007). We used strains harboring mutations in hrdl-1, hrd-1 and marc-6 genes to determine if these proteins are required for regulating spontaneous reversal behavior in C. elegans.Spontaneous reversals are a C. elegans behavior whose frequency is regulated by well-defined circuitry and neurotransmitter receptors, including the glutamate receptor, glr-1 (Brockie et al. 2001; Burbea et al. 2002; Dahlberg and Juo 2014, Hart et al. 1995; Kowalski et al. 2011; Zheng et al. 1999). We hypothesized that the spontaneous reversal frequency behavior of C. elegans would be affected by E3 ligase mutations if they are important for normal spontaneous reversal behavior. glr-1 animals reverse significantly less than wild-type animals and were used as a positive control (Figure 1B) (Hart et al. 1995; Kowalski et al. 2011). Animals harboring mutations in marc-6 and hrd-1 also reverse significantly less than wild-type animals (Figure 1A). Animals lacking full-length hrdl-1 reversed less than wild-type animals, but this was not statistically significant (Figure 1A).The primary motivation for our work was to ask if the glutamate receptor, GLR-1, might be regulated by these E3s, but further research must be done in order to determine molecular mechanisms that cause the differences in behavior that we report. Because ERAD E2 and E3 proteins can compensate for each others absence, we hypothesize that in the absence of any one E3 ligase, others are upregulated either through protein activity or gene expression (Bays et al. 2001; Weber et al. 2016). For example, if HRD-1 is upregulated to compensate for the putative loss-of-function of HRDL-1 in hrdl-1 mutant animals, this could explain why hrdl-1 animals did not show a statistically significant reduction in reversals/minute compared to wild-type animals. However, our results from the hrd-1 and marc-6 mutants suggest that there is not complete redundancy between the E3 ligases. Future experiments will focus on testing this hypothesis using double and triple mutants in the E3 ligase genes.

Sasagawa Y et al. (2007) Genes Cells "ER E3 ubiquitin ligase HRD-1 and its specific partner chaperone BiP play important roles in ERAD and developmental growth in Caenorhabditis elegans."

Yamanaka K, Ogura T, Sasagawa Y

[Genes Cells, 2007]

p97 (also called VCP or Cdc48p) and E3 ubiquitin ligases are the key players in retrotranslocation and ubiquitination of substrates in the endoplasmic reticulum-associated degradation (ERAD) pathways. Although their biochemical properties have been well studied, their cellular functions in development have not been revealed. Here, we investigate cellular functions of p97 and E3 ubiquitin ligases in Caenorhabditis elegans as a model organism. We found that C. elegans possesses three E3 ubiquitin ligases (named as HRD-1, HRDL-1 and MARC-6) like mammals, and that their simultaneous depletion caused extremely delayed growth. By monitoring the expression of an ER chaperone gene, it was revealed that p97 and HRD-1 play essential roles in unfolded protein response (UPR) and ERAD pathways. We further found that HRD-1 functions in concert with BiP, and that two BiP paralogues are functionally diversified. HRD-1 and BiP(HSP-3) play important roles in the developmental growth and function of intestinal cells, while HRD-1 and BiP(HSP-4) in the gonad formation. We propose that E3 ubiquitin ligases function in concert with a specific partner chaperone.

Coux O (2003) Biochem Soc Trans "An interaction map of proteasome subunits."

Coux O

[Biochem Soc Trans, 2003]

Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits (Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821-828). A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three alpha-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming 'interaction clusters' of

Oswald , Mackenzi et al. (2020) MicroPubl Biol

Dahlberg, Caroline (Lina), Oswald , Mackenzi, Hulsey-Vincent, Heino

[MicroPubl Biol, 2020]

Maintaining proteostasis, or protein homeostasis, is an important cellular function because misfolded proteins can aggregate and contribute to neurodegenerative diseases. One way that cells preserve proteostasis is through the Endoplasmic Reticulum Associated Degradation pathway (ERAD). ERAD relies on interactions between E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases to ubiquitylate misfolded proteins in order to signal for their destruction via the proteasome (Vembar and Brodsky 2008). We investigated if missense mutations in genes that encode two E2 ubiquitin-conjugating enzymes in C. elegans, UBC-6 and UBC-7, affect spontaneous reversal frequency (Brockie et al. 2001; Jones et al. 2002; Stewart et al. 2016; Zheng et al. 1999). UBC-6 and UBC-7 are conserved across Eukaryotes (Figure 1C). We obtained strains of animals from the Million Mutation Project with the missense mutations that confer the amino acid changes T158I in UBC-6 and P79L in UBC-7. The analogous residues in human Ube2j1 or Ube2j2 and Ube2g2 are Asp160 or 153 and Proline 78, respectively, which were mapped onto the available structures of the human enzymes (Figure 1C). In Ube2j2 (C. elegans UBC-6) Asp153 makes contact with Phe156, and mutating this residue decreases the efficiency of the enzyme (Tobi Ritterhoff, personal communication). Structural alignments suggest that Pro78 (mutated in UBC-7) is part of the well-conserved HPN (Histadine-Proline-Asparagine) motif, so mutation of the analogous residue was hypothesized to abrogate enzymatic activity (Cook and Shaw 2012; Wu et al. 2003).Our results show that the E2 ubiquitin-conjugating enzyme mutations did not significantly affect C. elegans spontaneous reversal frequency (Figure 1A). glr-1 animals were used as a control and reversed significantly less frequently than wild-type animals, as previously reported (Kowalski et al. 2011). Both ubc-6 and ubc-7 animals had less frequent spontaneous reversals than WT animals and more frequent spontaneous reversals than glr-1 animals, but neither of these results were significant.One clear reason for the insignificant change in reversal frequency in the ubc-6 and ubc-7 mutant animals could be that the missense mutations in these strains we observed do not affect gene function. It is also possible that UBC-6 and UBC-7 are able to compensate for each other in C. elegans. In S. cerevisiae Ubc6p and Ubc7p work cooperatively and sequentially to target substrates in coordination with the E3 ligases Doa10p (orthologous to C. elegans MARC-6), and Hrd1p (orthologous to C. elegans HRD-1 and paralogous to HRDL-1) (Sasagawa et al. 2007; Weber et al. 2016). It could be that this mechanism of tandem ubiquitylation is not conserved in C. elegans despite its conservation for human UBE2J2 (Weber et al. 2016). The human orthologs HRD1 and UBE2J1 (an additional ortholog of Ubc6p) also work together to target substrate proteins for degradation (Bays et al. 2001; Burr et al. 2011). Therefore it is possible that C. elegans E3s could collaborate with either UBC-6 or UBC-7 to target substrate proteins for degradation. In the future, we plan to address these possibilities using double ubc-6; ubc-7 point mutants and CRISPR-Cas9-targeted deletion mutants for UBC-6 and UBC-7, in combination with E3 ligase mutants.