Rab11-FIP3 binds dynein light intermediate chain 2 and its overexpression fragments the Golgi complex

The mechanochemical forces that move and position intracellular organelles and their intermediates in eukaryotic cells are provided by molecular motor proteins which include the cytoplasmic dynein-1 motor complex. Recently, we identified the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) as a novel binding-partner for dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1), a subunit of cytoplasmic dynein-1. Here, we show that FIP3 also binds the dynein light intermediate chain 2 subunit (DLIC-2, gene symbol DYNC1LI2). We show that like DLIC-1, DLIC-2 binds the amino-terminal 435 amino acids of FIP3 and that FIP3 links Rab11a to DLIC-2. We also show that FIP3 recruits DLIC-2 onto membranes and that DLIC-2 is necessary for the accumulation of endocytosed-transferrin (Tfn) at the pericentrosomal endosomal-recycling compartment (ERC). Finally, we demonstrate that overexpression of FIP3 fragments the Golgi complex by sequestering cytoplasmic dynein-1. In conclusion, we have identified FIP3 as the first membrane-associated interacting-partner for DLIC-2 and propose that this interaction serves to control endosomal trafficking from sorting endosomes to the ERC.     

Regulation of the NF-kappaB activation pathway by isolated domains of FIP3/IKKgamma, a component of the IkappaB-alpha kinase complex

FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is an essential component of the multimeric IkappaB-alpha kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-kappaB-inducing kinase, IKKbeta) of the NF-kappaB activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) alpha-induced NF-kappaB activity as well as to induce cell death when overexpressed. The adenovirus E3-14.7-kDa protein (E3-14.7K) is an inhibitor of TNFalpha-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions. The NF-kappaB inhibitory activity and the E3-14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNFalpha-induced IkappaB-alpha phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-kappaB underlies the FIP3 inhibition of NF-kappaB activity. The amino-terminal 119 amino acids were responsible for the FIP3-IKKbeta and FIP3-IKKalpha interaction, and the middle of the protein (amino acids 201-300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor-interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the IkappaB-alpha kinase complex. Whereas the full-length protein is required for efficient cell death, the amino-terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer.     

Ddi1-like protein from Leishmania major is an active aspartyl proteinase

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A₂ family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs.     

Crystal Structure of the C-Terminal Domain of Human DPY-30-Like Protein: A Component of the Histone Methyltransferase Complex

The conserved DPY-30 is an essential component of the dosage compensation complex that balances the X-linked gene expression by regulation of the complex formation in Caenorhabditis elegans. The human DPY-30-like protein (DPY-30L) homolog is a conserved member of certain histone methyltransferase (HMT) complexes. In the human MLL1 (mixed-lineage leukemia-1) HMT complex, DPY-30L binds to the BRE2 homolog ASH2L in order to regulate histone 3-lysine 4 trimethylation. We have determined the 1.2-Å crystal structure of the human DPY-30L C-terminal domain (DPY-30L_C). The DPY-30L_C structure, harboring the conserved DPY-30 motif, is composed of two α-helices linked by a sharp loop and forms a typical X-type four-helix bundle required for dimer formation. DPY-30L_C dimer formation is largely mediated by an extensive hydrophobic interface with some additional polar interactions. The oligomerization of DPY-30L_C in solution, together with its reported binding to ASH2L, leads us to propose that the hydrophobic surface of the dimer may provide a platform for interaction with ASH2L in the MLL1 HMT complex.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53^K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.     

Prediction of Protein-Protein Interfaces on G-Protein β Subunits Reveals a Novel Phospholipase C β2 Binding Domain

Gβ subunits from heterotrimeric G-proteins (guanine nucleotide-binding proteins) directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gβ. Although mammals contain five Gβ genes comprising two classes (Gβ1-like and Gβ5-like), plants and fungi have a single ortholog, and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gβ subunits and complementary biochemical data highlight specific sites within Gβs needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gβ molecular surface comprise interaction interfaces essential to Gβ's role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously unknown putative interfaces on the surface of Gβ. We show that one such novel interface occurs between Gβ and phospholipase C β2 (PLC-β2), a mammalian Gβ interacting protein. Substitutions of residues within this Gβ-PLC-β2 interface reduce the activation of PLC-β2 by Gβ1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gβ-protein interfaces. Similarly, we hypothesize that the seven remaining untested novel regions contribute to putative interfaces for other Gβ interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17?

The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.     

Parkin-mediated K63-linked polyubiquitination: A signal for targeting misfolded proteins to the aggresome-autophagy pathway

The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.     

Alternative polyadenylation trans-factor FIP1 exacerbates UUO/IRI-induced kidney injury and contributes to AKI-CKD transition via ROS-NLRP3 axis

NLRP3, a decisive role in inflammation regulation, is obviously upregulated by oxidative stress in kidney injury. The NLRP3 upregulation leads to unsolved inflammation and other pathological effects, contributing to aggravation of kidney injury and even transition to chronic kidney disease (CKD). However, the mechanism for NLRP3 upregulation and further aggravation of kidney injury remains largely elusive. In this study, we found NLRP3 3′UTR was shortened in response to kidney injury in vivo and oxidative stress in vitro. Functionally, such NLRP3 3′UTR shortening upregulated NLRP3 expression and amplified inflammation, fibrogenesis, ROS production and apoptosis, depending on stabilizing NLRP3 mRNA. Mechanistically, FIP1 was found to bind to pPAS of NLRP3 mRNA via its arginine-rich domain and to induce NLRP3 3′UTR shortening. In addition, FIP1 was upregulated in CKD specimens and negatively associated with renal function of CKD patients. More importantly, we found FIP1 was upregulated by oxidative stress and required for oxidative stress-induced NLRP3 upregulation, inflammation activation, cell damage and apoptosis. Finally, we proved that FIP1 silencing attenuated the inflammation activation, fibrogenesis, ROS production and apoptosis induced by UUO or IRI. Taken together, our results demonstrated that oxidative stress-upregulated FIP1 amplified inflammation, fibrogenesis, ROS production and apoptosis via inducing 3′UTR shortening of NLRP3, highlighting the importance of crosstalk between oxidative stress and alternative polyadenylation in AKI-CKD transition, as well as the therapeutic potential of FIP1 in kidney injury treatment.Subject terms: Acute kidney injury, Inflammasome     

An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis Fip1 homolog is characterized. The gene for this protein resides on chromosome V and encodes a 1196-amino acid polypeptide. Yeast two-hybrid and in vitro assays indicate that the N-terminal 137 amino acids of the Arabidopsis Fip1 protein interact with poly(A) polymerase (PAP). This domain also stimulates the activity of the PAP. Interestingly, this part of the Arabidopsis Fip1 interacts with Arabidopsis homologs of CstF77, CPSF30, CFIm-25, and PabN1. The interactions with CstF77, CPSF30, and CFIm-25 are reminiscent in various respects of similar interactions seen in yeast and mammals, although the part of the Arabidopsis Fip1 protein that participates in these interactions has no apparent counterpart in other eukaryotic Fip1 proteins. Interactions between Fip1 and PabN1 have not been reported in other systems; this may represent plant-specific associations. The C-terminal 789 amino acids of the Arabidopsis Fip1 protein were found to contain an RNA-binding domain; this domain correlated with an intact arginine-rich region and had a marked preference for poly(G) among the four homopolymers studied. These results indicate that the Arabidopsis Fip1, like its human counterpart, is an RNA-binding protein. Moreover, they provide conceptual links between PAP and several other Arabidopsis polyadenylation factor subunit homologs.     

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2-T1R3 Human Sweet Receptor

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.     

优雅蝈螽水溶性和类膜结合型海藻糖酶编码cDNA的鉴定及其在体内的表达

海藻糖酶是一种二糖水解酶,催化海藻糖转换为葡萄糖,为昆虫包括发育、壳多糖合成及飞翔代谢在内的多种生理过程所必需。尽管某些昆虫的海藻糖酶基因已被鉴定,但优雅蝈螽的海藻糖酶编码序列尚未见报告。本研究采用RACE结合多重PCR技术,分离鉴定优雅蝈螽的水溶性海藻糖酶（GgTre1）和类膜结合型海藻糖酶（GgTre2-like）的全长编码序列（cDNA）,包括携带不同长度3′-非翻译区（3′-UTR）的3个GgTre1 cDNA 亚型（GenBank：No.KY400001-KY400003）和3个GgTre2-like cDNA 亚型（GenBank：No.KY400004-KY400006）。 3个GgTre1 cDNA序列分别为2 107,2 021和1 914 bp,具有相同长度的5′-UTR（33 bp）,但3′-UTR 长度不同,分别为322,248和129 bp。GgTre1-2 cDNA含1 740 bp,编码579 个氨基酸残基组成的多肽链,分子量为67.29 kD;与之不同,根据cDNA演绎的GgTre1-1和GgTre1-3序列较GgTre1-2多4个氨基酸残基,多肽链的分子量为67.88 kD。3个GgTre2-like cDNA（GgTre2-like-1,-2和-3）序列全长分别为2 491,2 460 和2 381 bp。5′-UTR 均为284 bp,3′-UTR 分别为398,367和285 bp。GgTre2-like cDNA开阅读框为1 809 bp,编码602 氨基酸残基组成的多肽链,分子量为67.88 kD。实时定量PCR, 分析GgTre1和GgTre2-like基因在雌、雄个体（各20个）的组织特异性表达。结果显示,GgTre1 在卵巢和附腺表达量最高;GgTre2-like 主要在卵巢表达,在雄性肌肉和马氏管的表达量高于其他组织。上述结果表明,本研究从优雅蝈螽分离到3′-UTR长度不同的3个水溶性和3个类膜结合型海藻糖酶cDNA序列。结果还提示,GgTre1 在各组织的表达差异较大,而GgTre2-like 在各组织的表达相对稳定。不同长度3′-UTR的GgTre1 和 GgTre2-like 亚型的存在,以及不同长度的3′-UTR在翻译过程中的特殊作用,尚待今后研究证实。     

Complete nucleotide sequence of an unusual mobile element from trypanosoma brucei

The complete nucleotide sequence of a mobile element from Trypanosoma brucei is presented along with the sequence of its target site, which shows that the insertion has generated a 7 base pair direct repeat. The cloned copy of the element is a dimeric structure, one end of each monomer consisting of a stretch of 14 A residues preceded by a putative trypanosome polyadenylation signal. Six base pairs of DNA of unknown origin are found in the dimer between the two copies of the element. Evidence suggests that the element is present in the genome mainly as a monomer whose sequence is conserved across several species of trypanosome. The element contains an open reading frame encoding the same 160 amino acid protein in both sequenced copies and is extensively transcribed from both strands.     

Localization of serum resistance‐associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human‐infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance‐associated protein (SRA protein), protects against ApoL1‐mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA‐mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well‐characterized endosomal markers. By three‐dimensional deconvolved immunofluorescence single‐cell analysis, combined with double‐labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.     

Improving the resistance of a eukaryotic β-barrel protein to thermal and chemical perturbations

β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the “mitochondrial porin family” and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for β-barrel membrane proteins, we have identified three unfavorable β-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.