3.2.2. ER-Phagy
UFMylation has an important role in ER-phagy. Data from several recent studies have confirmed that UFMylation regulates ER degradation through lysosomes, thus furthering our understanding of the mechanisms by which UFMylation regulates ER stress. A human genome-wide screen indicated that UfBP1 is an ER-phagy regulator
[22][52]. Many factors associated with the ribosome and translational quality control, such as RPL26 and RPN1, were identified as UFMylation substrates that mediate ER-associated autophagy
[22,24,77][52][53][54]. NADH-cytochrome b5 reductase 3 (CYB5R3) is also UFMylated, and this mediates its degradation by lysosomes
[23][55].
CDK5RAP3 has been proposed to function as both a substrate adaptor that directs UFMylation toward target substrates, such as ribosomal protein RPL26
[47][26], and an adaptor protein for UFMylation-dependent ER-phagy
[23,65][47][55]. The results from one study showed that CDK5RAP3 depletion increased the amounts of GFP-UFM1-conjugated CYB5R3 and UfBP1, while treatment with bafilomycin A1 to suppress autophagy had no effect
[23][55]. These results support the idea that CDK5RAP3 promotes UFMylation-mediated ER-phagy.
The UFMylation of ribosomal proteins is believed to control the ribosomal stress response and ribosome-mediated protein translation quality. Indeed, UFL1 interacts with ribosomes, and the UFMylation substrate screening of ribosomal proteins showed that many ribosomal subunits are UFMylated
[78][56]. Similarly, UfBP1-dependent UFMylation substrate screening with nutrient starvation led to the identification of several ribosomal subunits, ribosome-associated factors, and ER-resident translocon proteins as UFMylation substrates, including RPL7A, RPLP0, RPL10A, RPL30, RPL19, and RPN1
[22][52]. These findings suggest that ribosomal proteins are likely modified by UFMylation, although most candidates remain to be confirmed experimentally.
RPL26 is UFMylated at K132 and K134
[24,77][53][54] when it is located at the ER surface, as the UFL1/CDK5RAP3/UfBP1 E3 complex is restricted to the ER membrane
[24][53]. This modification can be upregulated after treatment with the protein translation inhibitor anisomycin, indicating that ribosome arrest during cotranslational translocation in the ER is a specific trigger for RPL26 UFMylation
[77][54].
CYB5R3 is another master regulator of ER-phagy and is UFMylated at K214
[23][55] while it is anchored to the ER membrane. The researchers who used genome-wide CRISPR screening to identify UFL1 and UfBP1 as activators of ER-phagy
[22][52] explored whether CYB5R3 UFMylation is involved in this process. They showed that CYB5R3 contains FAD- and NADH-binding domains and catalyzes the transfer of reducing equivalents from NADH to cytochrome b5, which then acts as an electron donor
[80][57].
3.2.3. Autophagy
Although UFMylation regulates ER-phagy, the involvement of this PTM in general autophagy remains uncertain. Results from one study indicated that UFL1 deficiency impairs autophagy activity. LC3B associates with autophagosome development and maturation
[83][58] and p62/SQSTM1 serves as a bridge between LC3 and polyubiquitinated proteins, which are selectively packaged into autophagosomes. Therefore, LC3B and p62/SQSTM1 reflect the levels of autophagy
[84][59]. Indeed, UFL1 depletion in bone marrow (BM) cells resulted in increased ER stress and an increase in the abundance of LC3B and p62/SQSTM1, indicating that UFMylation regulates ER stress and general autophagy
[67][60]. However, the knockout of UfBP1 in BM cells did not influence the levels of LC3B or p62/SQSTM1
[19][30]. As UfBP1 normally functions synergistically with UFL1 to promote UFMylation, the opposite effects caused by defects in these two proteins seem contradictory. Interestingly, a genome-wide CRISPR screen of neuroglioma H4 cells identified several novel modulators of p62/SQSTM1, including the UFMylation cascade, which regulates p62/SQSTM1 expression by eliciting a cell-type-specific ER stress response, although few LC3B expression changes were evident when UFM1 was depleted
[85][61].
3.3. UFMylation and Development
UFMylation has an important impact on embryonic development. The complete depletion of UBA5 is embryonically lethal, with most UBA5
−/− mice embryos succumbing between 12.5 and 13.5 embryonic days (E12.5-E13.5) after gestation. By contrast, UBA5-heterozygous (UBA5
+/−) mice are born healthy and fertile without the emergence of any noticeable pathology for at least 2 years
[86][62]. Similarly, the complete depletion of UFL1 is embryonically lethal, with most UFL1
−/− mice embryos succumbing before E11.5, and as early as E10.5; UFL1
+/− mice are born healthy
[67][60]. UfBP1 depletion also causes death during embryonic development. While UfBP1
+/− mice are born healthy, most UfBP1
−/− mice embryos succumb by E12.5
[19][30]. Finally, CDK5RAP3 depletion also results in embryonic lethality by E8.5
[87][63].
3.3.1. Erythroid Development
An UFMylation deficiency causes the failed development of erythroid lineages
[19,67,86][30][60][62]. An analysis of
UBA5−/− mouse embryos at different developmental stages revealed a marked fetal anemia phenotype compared with
UBA5+/+ mouse embryos that was rescued by transgenic expression of UBA5 in the erythroid lineage
[86][62]. The loss of UFL1 blocked autophagic degradation and increased mitochondrial mass and ROS production in bone marrow cells, leading to the DNA damage response, p53 activation, and ER stress. This ER stress and the resulting generation of UPR enhanced hematopoietic stem cell death and impaired hematopoietic development, resulting in severe anemia, cytopenia, and ultimately animal death
[66][48].
3.3.2. Skeletal Development
Several studies have indicated the importance of UfBP1 in cartilage growth and development. Alongside this, reports that UfBP1 mutations are involved in spondylo-epi-metaphyseal dysplasia Shohat type (SEMDSH) disease indicate that UfBP1 has important roles in skeletal development. The whole-exome sequencing of four SEMDSH-prone families revealed a splice variant of UfBP1 (c.408 + 1G > A) resulting in a premature stop codon that causes a loss of function
[89][64]. Two unrelated SEMDSH patients were found to carry a different mutation of UfBP1 (G135K), which was associated with a similar phenotype to the UfBP1 (c.408 + 1G > A) mutation. These findings indicate that UfBP1 is associated with SEMDSH and skeletal development
[90][65]. In support of this association, UfBP1 depletion in zebrafish embryos resulted in craniofacial defects, and the deletion of UfBP1 in mouse embryos significantly increased limb bud apoptosis and cell death. Mechanistically, UfBP1 binds directly to SOX9, a major transcription factor for chondroblasts, to inhibit SOX9 ubiquitination and proteasomal degradation. COL2A1, the downstream target of SOX9, is linked to skeletal disorders. Therefore, UfBP1 defects lead to skeletal dysplasia by disturbing the SOX9-COL2A1 axis
[89][64].
3.3.3. Brain Development
Many studies have implicated UFMylation in brain development. Genetic studies have revealed that variants of the human
UBA5,
UFC1, and
UFM1 genes are associated with a number of neurodevelopmental diseases, including infantile-onset encephalopathy
[92][66], autosomal recessive cerebellar ataxia
[93][67], and microcephaly
[45][24]. Using exome sequencing, two groups found two biallelic mutations in
UBA5 (A371T and a loss-of-function nonsense mutation) that led to postnatal microcephaly, epilepsy, and spasticity in severe epileptic syndrome patients
[92,93,94][66][67][68]. CNS-specific knockout of UFM1 in mice caused neonatal death accompanied by microcephaly and the apoptosis of specific neurons
[92][66].
3.3.4. Development of Other Organs and Tissues
UFMylation is also involved in the development of other organs and tissues. Nephron-tubule-specific UFL1-KO mice presented kidney atrophy and interstitial fibrosis, demonstrating the crucial role of UFL1 in regulating kidney function
[95][69]. Hepatocyte-specific UFL1-KO induced hepatocyte apoptosis and mild steatosis in mice at 2 months of age and hepatocellular ballooning, extensive fibrosis, and steatohepatitis at 6–8 months of age
[96][70]. The deletion of
UFL1 in cardiomyocytes and intestinal epithelial cells caused heart failure and an increased susceptibility to experimentally-induced colitis, respectively, suggesting that UFL1 has an essential role in the maintenance of homeostasis in these organs
[97,98][71][72].
3.4. UFMylation and Immune Response
Recent work has shed light on the important role of UFL1 in antiviral innate immunity after DNA virus infection
[100][73]. UFL1 protein levels were significantly downregulated when peritoneal macrophages were infected with DNA viruses, such as the herpes simplex virus (HSV-1) or vaccinia virus (VACV), which also significantly decreased the mRNA expression of interferon β1, interleukin-6, and tumor necrosis factor. These results suggest that UFL1 promotes antiviral innate immunity. Further studies showed that UFL1 regulates the cGAS-STING pathway through its effects on STING stability. The E3 ligase TRIM29 ubiquitinates STING at K338/347/370, promoting its proteasome-dependent degradation
[101,102][74][75]. UFL1 competitively binds to STING to inhibit K48-linked ubiquitination, thereby maintaining STING protein stability and ultimately promoting antiviral innate immunity
[100][73].
3.5. UFMylation and Cancers
A comprehensive analysis of genomic alterations in the eight UFMylation family genes (
UFM1,
UBA5,
UFC1,
UFL1,
UfBP1,
CDK5RAP3,
UfSP1, and
UfSP2) across the TCGA database of 33 cancer types identified 55 recurrent and focal somatic copy number alteration events in UFMylation family genes
[105][76]. Among the UFMylation genes,
UfSP2 was frequently deleted in 14 cancer types. Calculations of the frequencies of copy number gain or loss for UFMylation genes in each cancer type revealed that
UfSP2 (31%),
UFM1 (31%), and
UFL1 (28%) had the highest average frequency of copy number loss, whereas
UFC1 (34%),
UfSP1 (34%), and
UfBP1 (30%) had the highest average frequency of copy number gain
[105][76]. In total, 11.08% of the TCGA samples had high-level copy number alterations in at least one of the eight genes
[105][76].