Prickle and Ror modulate Dishevelled-Vangl interaction to regulate non-canonical Wnt signaling during convergent extension.

Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and "core members" of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.

The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

AI-Based Protein Interaction Screening and Identification (AISID).

In this study, we presented an AISID method extending AlphaFold-Multimer's success in structure prediction towards identifying specific protein interactions with an optimized AISIDscore. The method was tested to identify the binding proteins in 18 human TNFSF (Tumor Necrosis Factor superfamily) members for each of 27 human TNFRSF (TNF receptor superfamily) members. For each TNFRSF member, we ranked the AISIDscore among the 18 TNFSF members. The correct pairing resulted in the highest AISIDscore for 13 out of 24 TNFRSF members which have known interactions with TNFSF members. Out of the 33 correct pairing between TNFSF and TNFRSF members, 28 pairs could be found in the top five (including 25 pairs in the top three) seats in the AISIDscore ranking. Surprisingly, the specific interactions between TNFSF10 (TNF-related apoptosis-inducing ligand, TRAIL) and its decoy receptors DcR1 and DcR2 gave the highest AISIDscore in the list, while the structures of DcR1 and DcR2 are unknown. The data strongly suggests that AlphaFold-Multimer might be a useful computational screening tool to find novel specific protein bindings. This AISID method may have broad applications in protein biochemistry, extending the application of AlphaFold far beyond structure predictions.

Protein kinase 2 (CK2) controls CD4+ T cell effector function in the pathogenesis of colitis.

Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4+ T-helper type 1 (Th1) and Th17 responses. Protein kinase 2 (CK2) is a conserved serine-threonine kinase involved in signal transduction pathways, which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3+ regulatory T cells. The function of CK2 in CD4+ T cells during the pathogenesis of CD is unknown. We utilized the T cell-induced colitis model, transferring CD45RBhi-naive CD4+ T cells from CK2αfl/fl controls and CK2αfl/fldLck-Cre mice into Rag1-/- mice. CD4+ T cells from CK2αfl/fldLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased interleukin-17A-positive (IL-17A+), interferon-γ-positive (IFN-γ+), and double-positive IL-17A+IFN-γ+ CD4+ T cells in the spleen and colon. We determined that CK2α regulates CD4+ T cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4+ T cell responses by regulating NFAT2, which is vital for T cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4+ T cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.

ß-amyloid redirects norepinephrine signaling to activate the pathogenic GSK3ß/tau cascade.

The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting ß-amyloid (Aß) and tau, two key pathological components of AD pathogenesis. Our results show that Aß oligomers bind to an allosteric site on α2A adrenergic receptor (α2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3ß (GSK3ß) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3ß/tau activation in response to nanomolar accumulations of extracellular Aß, which is 50- to 100-fold lower than the amount required to activate GSK3ß by Aß alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aß proteotoxicity, which might have strong implications for the interpretation of Aß clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD.

Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction.

The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.

Endoplasmic Reticulum Stress Signaling Pathways: Activation and Diseases.

Secretory and membrane proteins are folded in the endoplasmic reticulum (ER) prior to their exit. When ER function is disturbed by exogenous and endogenous factors, such as heat shock, ultraviolet radiation, hypoxia, or hypoglycemia, the misfolded proteins may accumulate, promoting ER stress. To rescue this unfavorable situation, the unfolded protein response is activated to reduce misfolded proteins within the ER. Upon ER stress, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6, are activated. Here, we discuss the mechanisms of PERK and IRE1 activation and describe two working models for ER stress initiation: the BiP-dependent model and the ligand-driven model. ER stress activation has been linked to multiple diseases, including cancers, Alzheimer's disease, and diabetes. Thus, the regulation of ER stress may provide potential therapeutic targets for these diseases.

The luminal domain of the ER stress sensor protein PERK binds misfolded proteins and thereby triggers PERK oligomerization.

PRKR-like endoplasmic reticulum kinase (PERK) is one of the major sensor proteins that detect protein folding imbalances during endoplasmic reticulum (ER) stress. However, it remains unclear how ER stress activates PERK to initiate a downstream unfolded protein response (UPR). Here, we found that PERK's luminal domain can recognize and selectively interact with misfolded proteins but not with native proteins. Screening a phage-display library, we identified a peptide substrate, P16, of the PERK luminal domain and confirmed that P16 efficiently competes with misfolded proteins for binding this domain. To unravel the mechanism by which the PERK luminal domain interacts with misfolded proteins, we determined the crystal structure of the bovine PERK luminal domain complexed with P16 to 2.8-Å resolution. The structure revealed that PERK's luminal domain binds the peptide through a conserved hydrophobic groove. Substitutions within hydrophobic regions of the PERK luminal domain abolished the binding between PERK and misfolded proteins. We also noted that peptide binding results in major conformational changes in the PERK luminal domain that may favor PERK oligomerization. The structure of the PERK luminal domain-P16 complex suggested stacking of the luminal domain that leads to PERK oligomerization and activation via autophosphorylation after ligand binding. Collectively, our structural and biochemical results strongly support a ligand-driven model in which the PERK luminal domain interacts directly with misfolded proteins to induce PERK oligomerization and activation, resulting in ER stress signaling and the UPR.

Crystal structures of Hsp104 N-terminal domains from Saccharomyces cerevisiae and Candida albicans suggest the mechanism for the function of Hsp104 in dissolving prions.

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs from Saccharomyces cerevisiae (ScHsp104NTD) and Candida albicans (CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around the putative peptide-binding groove mediate the monomer-monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.

The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins.

PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Šresolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the ß-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible ß-sandwich domain to recognize and interact with a broad range of misfolded proteins.

The structure of Tim50(164-361) suggests the mechanism by which Tim50 receives mitochondrial presequences.

Mitochondrial preproteins are transported through the translocase of the outer membrane (TOM) complex. Tim50 and Tim23 then transfer preproteins with N-terminal targeting presequences through the intermembrane space (IMS) across the inner membrane. The crystal structure of the IMS domain of Tim50 [Tim50(164-361)] has previously been determined to 1.83 Šresolution. Here, the crystal structure of Tim50(164-361) at 2.67 Šresolution that was crystallized using a different condition is reported. Compared with the previously determined Tim50(164-361) structure, significant conformational changes occur within the protruding ß-hairpin of Tim50 and the nearby helix A2. These findings indicate that the IMS domain of Tim50 exhibits significant structural plasticity within the putative presequence-binding groove, which may play important roles in the function of Tim50 as a receptor protein in the TIM complex that interacts with the presequence and multiple other proteins. More interestingly, the crystal packing indicates that helix A1 from the neighboring monomer docks into the putative presequence-binding groove of Tim50(164-361), which may mimic the scenario of Tim50 and the presequence complex. Tim50 may recognize and bind the presequence helix by utilizing the inner side of the protruding ß-hairpin through hydrophobic interactions. Therefore, the protruding ß-hairpin of Tim50 may play critical roles in receiving the presequence and recruiting Tim23 for subsequent protein translocations.

Structural analysis of the Sil1-Bip complex reveals the mechanism for Sil1 to function as a nucleotide-exchange factor.

Sil1 functions as a NEF (nucleotide-exchange factor) for the ER (endoplasmic reticulum) Hsp70 (heat-shock protein of 70 kDa) Bip in eukaryotic cells. Sil1 may catalyse the ADP release from Bip by interacting directly with the ATPase domain of Bip. In the present study we show the complex crystal structure of the yeast Bip and the NEF Sil1 at the resolution of 2.3 Å (1 Å=0.1 nm). In the Sil1-Bip complex structure, the Sil1 molecule acts as a 'clamp' which binds lobe IIb of the Bip ATPase domain. The binding of Sil1 causes the rotation of lobe IIb ~ 13.5° away from the ADP-binding pocket. The complex formation also induces lobe Ib to swing in the opposite direction by ~ 3.7°. These conformational changes open up the nucleotide-binding pocket in the Bip ATPase domain and disrupt the hydrogen bonds between Bip and bound ADP, which may catalyse ADP release. Mutation of the Sil1 residues involved in binding the Bip ATPase domain compromise the binding affinity of Sil1 to Bip, and these Sil1 mutants also abolish the ability to stimulate the ATPase activity of Bip.

Structural basis for the function of Tim50 in the mitochondrial presequence translocase.

Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding ß-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.

The structure of the PERK kinase domain suggests the mechanism for its activation.

The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK's N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK's kinase domain has been determined to 2.8 Šresolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a `line-up' model for PERK activation triggered by oligomerization of its luminal domain.

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway.

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

Membrane binding mechanism of yeast mitochondrial peripheral membrane protein TIM44.

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.

Structural insight into the protective role of P58(IPK) during unfolded protein response.

P58(IPK) has been identified as an ER molecular chaperone to maintain protein-folding homeostasis. P58(IPK) expression can be significantly upregulated during unfolded protein responses (UPR), and it may play important roles in suppressing the ER protein aggregations. To investigate the mechanism how P58(IPK) functions to promote protein folding within ER, we have determined the crystal structure of P58(IPK) TPR domain at 2.5Å resolution. P58(IPK) contains nine TPR motifs and a C-terminal J domain within its primary sequence. The crystal structure of P58(IPK) revealed three subdomains (I, II, and III) with similar folds and each domain contains three TPR motifs. Our data also showed that P58(IPK) acts as a molecular chaperone by interacting with the unfolded proteins such as luciferase, rhodanese, and insulin. The P58(IPK) structure reveals a conserved hydrophobic patch located in subdomain I that may be involved in binding the misfolded polypeptides. We have proposed a working model for P58(IPK) to act together with Bip to prevent protein aggregations and promote protein foldings within ER.

The structural plasticity of Tom71 for mitochondrial precursor translocations.

Mitochondrial precursors are transported through the translocase of the outer membrane (TOM) complex. Tom70/Tom71 is a major surface receptor of the TOM complex for mitochondrial precursors and facilitates Hsp70/Hsp90-escorted precursor translocation into the mitochondrion. Previous structural studies of Tom71 have revealed that it contains an N-terminal and a C-terminal domain and that the two domains may remain in an open conformation when binding to Hsp70/Hsp90. In a newly obtained crystal form of a complex of Tom71 and the Hsp70 C-terminus, the N-terminal domain was found to have rotated about 12 degrees towards the C-terminal domain compared with the previous determined crystal structure of Tom71 in the open conformation. This newly solved structure is defined as the ;intermediate conformation'. The domain rearrangements in Tom71 significantly change the surface hydrophobicity and the volume of the precursor-binding pocket. This work suggests that Tom70/Tom71-family members may exhibit structural plasticity from the intermediate conformation to the fully open conformation when complexed with Hsp70/Hsp90. This structural plasticity enables the precursor receptors to accommodate different precursor substrates for mitochondrial translocation.

Recognition of histone H3K4 trimethylation by the plant homeodomain of PHF2 modulates histone demethylation.

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications.

Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR.

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 A resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).