Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites.

Ubiquitin (Ub) shares the highest sequence identity with neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) in the Ub-like protein family. However, different enzyme systems are precisely employed for targeting Ub and NEDD8 to specific substrates. The molecular determinants for distinguishing between Ub and NEDD8 by Ub-specific peptidases (USPs) remain poorly characterized. By replacing the non-conserved residues of Ub with their NEDD8 equivalents by mutagenesis, and vice versa, we observed that the Ub4K, Ub12E, and Ub14E mutants partially and the Ub4K/12E/14E/72A mutant completely prevented their hydrolysis by USP2. The NEDD84F and NEDD814T mutants were slightly hydrolyzed by USP2; however, the NEDD812T/14T/72R and NEDD84F/12T/14T/72R mutants were accessible for hydrolysis by USP2, suggesting that Ub and NEDD8 residues 4, 12, 14, and 72 serve as the molecular determinants for specific recognition by USP2. We also demonstrated that the level of inhibition caused by Ub mutants with multiple mutation sites was not purely additive when compared with the single mutation results. Furthermore, USP2 was determined to bind to the N-terminus of Ub to form a stable interaction, after which it binds with the C-terminus of Ub to ensure substrate specificity. The same results were also discovered when Ub, Ub4K/12E/14E/72A, NEDD8, and NEDD84F/12T/14T/72R were incubated with USP21.

Dynamic modification of target proteins by small ubiquitin-like modifier (SUMO) is known to modulate many important cellular processes and is required for cell viability and development in all eukaryotes. However, understanding of SUMO systems in plants, especially in unicellular green algae, remains elusive. In this study, Chlamydomonas reinhardtii CrSUMO96, CrSUMO97 and CrSUMO148 were characterized. We show that the formation of polymeric CrSUMO96 and CrSUMO97 chains can be catalyzed either by the human SAE1/SAE2 and Ubc9 SUMOylation system in vitro or by an Escherichia coli chimeric SUMOylation system in vivo. An exposed C-terminal di-glycine motif of CrSUMO96 or CrSUMO97 is essential for functional SUMOylation. The human SUMO-specific protease, SENP1, demonstrates more processing activity for CrSUMO97 than for CrSUMO96. The CrSUMO148 precursor notably has four repeated di-glycine motifs at the C-terminus. This unique feature is not found in other known SUMO proteins. Interestingly, only 83-residual CrSUMO148(1-83) with the first di-glycine motif can form SAE1/SAE2-SUMO complex and further form polymeric chains with the help of Ubc9. More surprisingly, CrSUMO148 precursor is digested by SENP1, solely at the peptide bond after the first di-glycine motif although there are four theoretically identical processing sites in the primary sequence. This process directly generates 83-residual CrSUMO148(1-83) mature protein, which is exactly the form suitable for activation and conjugation. We also show that SENP1 displays similar isopeptidase activity in the deconjugation of polymeric CrSUMO96, CrSUMO97 or CrSUMO148 chains, revealing that the catalytic mechanisms of processing and deconjugation of CrSUMOs by SENP1 may differ.

The polytype structural variations of a set of SiC bulk wafers with different Nitrogen (N) doping levels, prepared by Physical Vapore Deposition (PVD), are studied. The initial growth conditions were used to produce 6H-polytype SiC, which has been approved for the undoped and lightly doped materials. However, when extreme high N-dopants were applied, the obtained wafer was found with 4H- and 15R-polytype features. Our experimental results of HR-TEM and Raman scattering have revealed clearly the polytype transformation, indicating that the inducement of N in the reactor leads to the polytype transformation of the resulted SiC crystal.

Ubiquitin‐like proteins (Ubls) share very high sequence and structural similarity; but conjugation to a protein leads to distinct biological consequences. Ubls are initially synthesized as precursor forms for processing by Ubl‐specific proteases to expose the C‐terminal di‐glycine motif for conjugation to target proteins. Ubiquitin and NEDD8 share 80% sequence identity, however, each exclusive protease can specifically react with ubiquitin or NEDD8. This work is aimed to study the substrate recognition mechanism of USP (ubiquitin‐specific protease) protein family.The crystal structure of USP2‐ubiquitin binding complex showed that residues 4, 12, 14 and 72 of ubiquitin were the major interaction sites. It is also noted that these residues are not conserved in NEDD8. By mutating these sites to the corresponding residues in NEDD8, we found that F4K ubiquitin mutant markedly inhibited its hydrolysis by USP2, and T12E and T14E ubiquitin mutants also showed partial catalytic inhibition due to the steric hindrance and electric repulsion between the mutated residue and USP2. Moreover, mutation of residue 72 further inhibited the hydrolysis of T12E/T14E/R72A ubiquitin mutant than T12E/T14E ubiquitin mutant. Because the key amino acids for interacting with residues 4, 12, 14 and 72 of ubiquitin are conserved in the USP protein family, this study suggests a general recognition mechanism by which USP can specifically discriminate between ubiquitin and NEDD8.