CFL-1, a novel F-box protein with leucine-rich repeat may interact with UNC-10 for the regulation of defecation and daumone response in Caenorhabditis elegans

Previously we reported that CFL-1, the single LRR-type F-box protein in the Caenorhabditis elegans genome, affected defecation behavior and daumone response. CFL-1 is highly homologous to the FBXL20 in mammals, which regulates synaptic vesicle release by targeting its substrate Rim1 for ubiquitin-mediated degradation. The worm homolog of Rim1 is UNC-10, a presynaptic membrane protein that triggers synaptic vesicle fusion through interaction with RAB-3 GTPase. To examine if CFL-1 exerts its modulatory effect on the defecation and daumone response via ubiquitination of UNC-10, we performed RNAi knock-down of CFL-1 in the unc-10(e102) mutant background. We noticed additive increase in defecation interval when the activities of both CFL-1 and UNC-10 were compromised. Also, the degree of dauer formation upon daumone treatment in unc-10 mutants treated with CFL-1 RNAi decreased further than the level observed in untreated mutants or wild type N2 worms with CFL-1 RNAi knock-down. Our data suggest that CFL-1 affects defecation frequency and daumone response in C. elegans through the ubiquitination of UNC-10.     

Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation

Deleted in Split hand/Split foot 1 (DSS1) was previously identified as a novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH(2) terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.     

Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation

Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.     

Structural basis for the interaction between human Npl4 and Npl4-binding motif of human Ufd1

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Two C-terminal ankyrin repeats form the minimal stable unit of the ankyrin repeat protein p18INK4c

Ankyrin repeat proteins (ARPs) appear to be abundant in organisms from all phyla, and play critical regulatory roles, mediating specific interactions with target biomolecules and thus ordering the sequence of events in diverse cellular processes. ARPs possess a non-globular scaffold consisting of repeating motifs named ankyrin (ANK) repeats, which stack on each other. The modular architecture of ARPs provides a new paradigm for understanding protein stability and folding mechanisms. In the present study, the stability of various C-terminal fragments of the ARP p18^INK4c was investigated by all-atomic 450 ns molecular dynamics (MD) simulations in explicit water solvent. Only motifs with at least two ANK repeats made stable systems in the available timescale. All smaller fragments were unstable, readily losing their native fold and α-helical content. Since each non-terminal ANK repeat has two hydrophobic sides, we may hypothesize that at least one hydrophobic side must be fully covered and shielded from the water as a necessary, but not sufficient, condition to maintain ANK repeat stability. Consequently, at least two ANK repeats are required to make a stable ARP. Figure Structure of the p18INK4c protein (PDB entry 1IHB, chain B), which is a member of the cyclin-dependent kinase inhibitor (INK) tumor suppressor family with five ankyrin (ANK) repeat modules. The figure was generated by PyMol Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

C-terminal domain of p16(INK4a) is adequate in inducing cell cycle arrest, growth inhibition and CDK4/6 interaction similar to the full length protein in HT-1080 fibrosarcoma cells

The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

Overexpression of Sna3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1

Tryptophan permease Tat2 in Saccharomyces cerevisiae undergoes Rsp5-dependent degradation upon exposure to high hydrostatic pressure and it limits the growth of tryptophan auxotrophs. Overexpression of SNA3 encoding an endosomal/vacuolar protein possessing the PPAY motif allowed growth at 25 MPa, which was potentiated by marked stabilization of Tat2. This appeared to depend on the PPAY motif, which interacted with the WW domain of Rsp5. Subcellular localization of Rsp5 was unchanged by overexpression of either SNA3 or SNA3-AAAY. While the loss of Bul1, a binding protein of Rsp5, or the rsp5-ww3 mutation allowed high-pressure growth, overexpression of BUL1 abolished the Sna3-mediated growth at 25 MPa. These results suggest that Sna3 and Bul1 compete for the WW domain of Rsp5 upon Tat2 ubiquitination.

Structured summary

MINT-7303515:PEP12 (uniprotkb:P32854) and TAT2 (uniprotkb:P38967) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK

WNK kinases have rapidly emerged as important regulators of Na⁺ and K⁺ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K⁺ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordon’s syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (ΔAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, ΔAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.     

Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate.

The human immunodeficiency virus type 1 integrase protein can be specifically cross-linked to viral long terminal repeat substrate oligonucleotides in vitro by using UV light. Site-directed mutagenesis and deletion analyses were used to define the domains involved in the interaction of integrase with the viral DNA substrate. Our results showed that mutation of conserved residues Pro-109 and Asp-116, which are found to be critical for the endonuclease and integration activities of IN protein, abolished the ability of the protein to cross-link to its DNA substrate. Furthermore, deletion analysis experiments showed that removal of 39 amino acids from the amino terminus and deletion of 15 amino acids from the carboxyl terminus abolished DNA cross-linking.     

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

Background

HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Starch-binding domain-containing protein 1 (Stbd1) and glycogen metabolism: Identification of the Atg8 family interacting motif (AIM) in Stbd1 required for interaction with GABARAPL1

Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. An important pathway for its degradation is by transport to lysosomes in an autophagy-like process. It has been proposed that starch-binding domain-containing protein 1 (Stbd1) may participate in this mechanism by anchoring glycogen to intracellular membranes. In addition, Stbd1 has been reported to interact with a known autophagy protein, GABARAPL1, a member of the Atg8 family. Here, we confirm this interaction and identify an Atg8 interacting motif (AIM) in Stbd1 necessary for GABARAPL1 binding as judged by co-immunoprecipitation from cell extracts and co-localization in cells as evidenced by immunofluorescence microscopy. The AIM sequence of Stbd1 ²⁰⁰HEEWEMV²⁰⁶ lies within a predicted disordered region of the molecule and fits the consensus of other AIM sequences in cargo-specifying proteins such as p62 and Nix. Mutation of the AIM, including single point mutations of either W203 or V206, eliminated the co-localization of Stbd1 with both over-expressed and endogenous GABARAPL1. Stbd1 may therefore function as a novel cargo binding protein that delivers glycogen to lysosomes in an autophagic pathway that could be termed “glycophagy”.