The integrity and organization of the human AIPL1 functional domains is critical for its role as a HSP90-dependent co-chaperone for rod PDE6

Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) are associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy in early childhood. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. In this study, we characterized the functional deficits of AIPL1 variations, some of which induce aberrant pre-mRNA AIPL1 splicing leading to the production of al- ternative AIPL1 isoforms. We investigated the ability of the AIPL1 variants to mediate an interaction with HSP90 and modulate the rod cGMP PDE6 stability and activity. Our data revealed that both the FK506 binding protein (FKBP)-like domain and the tetra- tricopeptide repeat (TPR) domain of AIPL1 are required for interaction with HSP90. We further demonstrate that AIPL1 signifi- cantly modulates the catalytic activity of heterologously expressed rod PDE6. Although the N-terminal FKBP-like domain of AIPL1 binds the farnesylated PDE6a subunit through direct interaction with the farnesyl moiety, mutations compromising the integrity of the C-terminal TPR domain of AIPL1 also failed to modulate PDE6 activity efficiently. These AIPL1 variants moreover failed to promote the HSP90-dependent stabilization of the PDE6a subunit in the cytosol. In summary, we have successfully vali- dated the disease-causing status of the AIPL1 variations in vitro. Our findings provide insight into the mechanism underlying the co-chaperone role of AIPL1 and will be critical for ensuring an early and effective diagnosis of AIPL1 LCA patients

Calcineurin B in Dictyostelium discoideum

The genome of Dictyostelium discoideum contains a single gene (cnb1) for the regulatory (B) subunit of the Ca2+/calmodulin dependent protein phosphatase, calcineurin. Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher Mr protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5´RACE amplification of the smaller transcript yielded cDNAs lacking the 5´ nontranslated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnb1 transcript is processed by an unconventional mechanism that leads to truncation of the 5´ untranslated region and at least the first AUG initiator codon and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnb1 gene.A truncated CNB cDNA fragment was recombinantly expressed in E. coli and purified. The purified protein was used for the production of polyclonal antibodies and for the biochemical studies. The biochemical datas optained are very similar to datas described for CN from other organisms. It was shown, that the recombinant CNB binds to CNA from Dictyostelium and that it increases the phosphatase activity of CNA using the RII peptide as a substrate but not when using pNPP

The ubiquitin-like modifier FAT10 in cancer development

During the last years it has emerged that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 expression is highly up-regulated by proinflammatory cytokines IFN-γ and TNF-α in all cell types and tissues and it was also found to be upregulated in many cancer types such as glioma, colorectal, liver or gastric cancer. While proinflammatory cytokines within the tumor microenvironment probably contribute to FAT10 overexpression, an increasing body of evidence argues that pro-malignant capacities of FAT10 itself largely underlie its broad and intense overexpression in tumor tissues. FAT10 thereby regulates pathways involved in cancer development such as the NF-κB- or Wnt-signaling. Moreover, FAT10 directly interacts with and influences downstream targets such as p53 or β-catenin, leading to enhanced survival, proliferation, invasion and metastasis formation of cancer cells but also of non-malignant cells. In this review we will provide an overview of the regulation of FAT10 expression as well as its function in carcinogenesis.publishe

The ubiquitin-like modifier FAT10 : much more than a proteasome-targeting signal

Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) also called ubiquitin D (UBD) is a member of the ubiquitin-like modifier (ULM) family. The FAT10 gene is localized in the MHC class I locus and FAT10 protein expression is mainly restricted to cells and organs of the immune system. In all other cell types and tissues, FAT10 expression is highly inducible by the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF). Besides ubiquitin, FAT10 is the only ULM which directly targets its substrates for degradation by the 26S proteasome. This poses the question as to why two ULMs sharing the proteasome-targeting function have evolved and how they differ from each other. This Review summarizes the current knowledge of the special structure of FAT10 and highlights its differences from ubiquitin. We discuss how these differences might result in differential outcomes concerning proteasomal degradation mechanisms and non-covalent target interactions. Moreover, recent insights about the structural and functional impact of FAT10 interacting with specific non-covalent interaction partners are reviewed.publishe

FAT10ylation as a signal for proteasomal degradation

The Nobel prize has been awarded for the discovery of ubiquitin as a transferable signal for the degradation of proteins by the 26S proteasome. While isopeptide linkage of a protein with a single ubiquitin does not serve as a degradation signal for the proteasome, poly-ubiquitylation via several different lysine residues within ubiquitin leads to efficient proteasomal degradation. Ubiquitin-like modifiers have not been shown to directly mediate proteasomal degradation except for the cytokine inducible modifier HLA-F adjacent transcript 10 (FAT10), which consists of two ubiquitin-like domains. FAT10 ends with a free diglycine motif at its C-terminus which is required for isopeptide linkage to hundreds of different substrates. In contrast to ubiquitin, a single FAT10 suffices to bind to the 26S proteasome and to efficiently mediate proteasomal degradation in a ubiquitin-independent manner. Here we review the data on ubiquitin-independent degradation by FAT10, on how FAT10 is conjugated to its substrates, how FAT10 binds to the 26S proteasome, and how the ubiquitin-like (UBL)-ubiquitin-associated (UBA) protein NUB1L accelerates FAT10 mediated proteolysis. Finally, with a glimpse on recently identified substrates, we will discuss the currently emerging knowledge about the biological functions of FAT10. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System

Investigations into the auto-FAT10ylation of the bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1

The cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) targets its substrates for degradation by the proteasome. FAT10 is conjugated to its substrates via the bispecific, ubiquitin-activating and FAT10-activating enzyme UBA6, the likewise bispecific conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By MS analysis, we found that USE1 undergoes self-FAT10ylation in cis, mainly at Lys323. Mutation of Lys323 to an arginine did not abolish auto-FAT10ylation of USE1, but every other lysine could instead be modified with FAT10. Similarly to bulk FAT10 substrates, FAT10ylation of USE1 accelerated its proteasomal degradation. Interestingly, the USE1–FAT10 conjugate continued to be an active E2 enzyme, because both FAT10 and ubiquitin could still be thioester-linked to the USE1–FAT10 conjugate. We therefore suggest that the major function of USE1 auto-FAT10ylation is to serve as a negative feedback mechanism to limit the conjugation of FAT10 upon its cytokine-mediated induction by reducing the amount of USE1 through proteasomal degradation of the USE1–FAT10 conjugate

Redox regulation of CD21 shedding involves signaling via PKC and indicates the formation of a juxtamembrane stalk

Soluble CD21 (sCD21), released from the plasma membrane by proteolytic cleavage (shedding) of its extracellular domain (ectodomain) blocks B cell/follicular dendritic cell interaction and activates monocytes. We show here that both serine- and metalloproteases are involved in CD21 shedding. Using the oxidant pervanadate to mimic B cell receptor activation and thiol antioxidants such as N-acetylcysteine (NAC) and glutathione (GSH) we show that CD21 shedding is a redox-regulated process inducible by oxidation presumably through activation of a tyrosine kinase-mediated signal pathway involving protein kinase C (PKC), and by reducing agents that either directly activate the metalloprotease and/or modify intramolecular disulfide bridges within CD21 and thereby facilitate access to the cleavage site. Lack of short consensus repeat 16 (SCR16) abolishes CD21 shedding, and opening of the disulfide bridge between cys-2 (Cys941) and cys-4 (Cys968) of SCR16 is a prerequisite for CD21 shedding. Replacing these cysteines with selenocysteines (thereby changing the redox potential from -180 to -381 mV) results in a loss of inducible CD21 shedding, and removing this bridge by exchanging these cysteines with methionines increases CD21 shedding

Newly translated proteins are substrates for ubiquitin, ISG15, and FAT10

The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins.publishe

Conjugation of the Ubiquitin Activating Enzyme UBE1 with the Ubiquitin-Like Modifier FAT10 Targets It for Proteasomal Degradation

<div><p>The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.</p></div

UBE1 is a substrate of FAT10ylation.

<p>(A) HEK293 cells were transiently transfected with expression plasmids for HA-UBE1, the active site cysteine mutant of HA-UBE1 (HA-UBE1 C632A), His-3xFLAG-FAT10 (FLAG-FAT10), His-3xFLAG-FAT10 with a mutated diglycine motif at the C terminus (FLAG-FAT10 ΔGG), or a lysine-less mutant of His-3xFLAG-FAT10 (FLAG-FAT10 K0). Where indicated, cells were additionally treated with 10 μM of the proteasome inhibitor MG132 for six hours prior to harvesting. Cell lysates were subjected to immunoprecipitation with anti-HA antibody coupled to agarose. Proteins were separated on 4–12% Bis/Tris NuPAGE gels, and western blot analysis was performed with antibodies reactive against HA or FLAG. β-actin was used as loading control. The upper panels show the immunoprecipitated proteins and the lower western blot panels show the total protein expression in the cell lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were transfected with the expression constructs indicated, harvested and lysed as described in (A). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody coupled to agarose and subsequent western blots were performed as described in (A). An asterisk in lane 6 marks an unspecific background band in the WB against FLAG. Cartoons describe the covalent and non-covalent interactions of UBE1 and FAT10. One representative experiment out of three experiments with similar outcomes is shown.</p