Motor domain-mediated autoinhibition dictates axonal transport by the kinesin UNC-104/KIF1A.

The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated in vivo is not fully understood. Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.

Structural Basis of the Differential Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy.

Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.

Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis.

Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural 'hinge' common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.

Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition.

In kinesin-3, the coiled-coil 1 (CC1) can sequester the preceding neck coil (NC) for autoinhibition, but the underlying mechanism is poorly understood. Here, we determined the structures of the uninhibited motor domain (MD)-NC dimer and inhibited MD-NC-CC1 monomer of kinesin-3 KIF13B. In the MD-NC-CC1 monomer, CC1 is broken into two short helices that unexpectedly interact with both the NC and the MD. Compared with the MD-NC dimer, the CC1-mediated integration of NC and MD not only blocks the NC dimer formation, but also prevents the neck linker (NL) undocking and the ADP release from the MD. Mutations of the essential residues in the interdomain interaction interface in the MD-NC-CC1 monomer restored the MD activity. Thus, CC1 fastens the neck domain and MD and inhibits both NC and NL. This CC1-mediated lockdown of the entire neck domain may represent a paradigm for kinesin autoinhibition that could be applicable to other kinesin-3 motors.

Calcium-induced structural rearrangements release autoinhibition in the Rap-GEF CalDAG-GEFI.

Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B. Structural differences between active (WT) and inactive (EF hand variant) CalDAG-GEFI protein were determined by hydrogen-deuterium exchange MS. The highest differential rates of deuterium uptake in WT over EF hand variant CalDAG-GEFI were observed in regions within the catalytic Cdc25 domain and a putative autoinhibitory linker connecting the Cdc25 and EF hand domains. Exchange activity in the EF hand variant was fully restored by an additional substitution, valine 406 to glutamate, which is thought to disrupt the interface between the autoinhibitory linker and the Cdc25 domain. Overall, our results suggest a model for how CalDAG-GEFI remains in an autoinhibited state when levels of cytosolic calcium in resting platelets are low. In response to cellular stimulation, calcium mobilization and binding to the EF hands causes conformational rearrangements within CalDAG-GEFI, including the autoinhibitory linker that frees the catalytic surface of CalDAG-GEFI to engage and activate Rap1B. The data from this study are the first evidence linking CalDAG-GEFI activity directly to calcium.

Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling.

Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling.

Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A.

Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3.

A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation.

The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf-MEK-ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2.

Interdomain interface-mediated target recognition by the Scribble PDZ34 supramodule.

Tandem-arranged PDZ [PSD-95 (postsynaptic density-95), Dlg (discs large homologue) and ZO-1 (zonula occludens-1)] domains often form structural and functional supramodules with distinct target-binding properties. In the present study, we found that the two PDZ domains within the PDZ34 tandem of Scribble, a cell polarity regulator, tightly pack in a 'front-to-back' mode to form a compact supramodule. Although PDZ4 contains a distorted αB/ßB pocket, the attachment of PDZ4 to PDZ3 generates an unexpected interdomain pocket that is adjacent to and integrates with the canonical αB/ßB pocket of PDZ3 to form an expanded target-binding groove. The structure of the PDZ34-target peptide complex further demonstrated that the peptide binds to this expanded target-binding groove with its upstream residues anchoring into the interdomain pocket directly. Mutations of the interdomain pocket and disruptions of the PDZ34 supramodule both interfere with its target-binding capacity. Therefore, the interdomain interface between the PDZ34 supramodule is intrinsically required for its target recognition and determines its target-binding specificity. This interdomain interface-mediated specific recognition may represent a novel mode of target recognition and would broaden the target-binding versatility for PDZ supramodules. The supramodular nature and target recognition mode of the PDZ34 tandem found in the present study would also help to identify the new binding partners of Scribble and thus may direct further research on the PDZ domain-mediated assembly of Scribble polarity complexes.

Brain tumor regulates neuromuscular synapse growth and endocytosis in Drosophila by suppressing mad expression.

The precise regulation of synaptic growth is critical for the proper formation and plasticity of functional neural circuits. Identification and characterization of factors that regulate synaptic growth and function have been under intensive investigation. Here we report that brain tumor (brat), which was identified as a translational repressor in multiple biological processes, plays a crucial role at Drosophila neuromuscular junction (NMJ) synapses. Immunohistochemical analysis demonstrated that brat mutants exhibited synaptic overgrowth characterized by excess satellite boutons at NMJ terminals, whereas electron microscopy revealed increased synaptic vesicle size but reduced density at active zones compared with wild-types. Spontaneous miniature excitatory junctional potential amplitudes were larger and evoked quantal content was lower at brat mutant NMJs. In agreement with the morphological and physiological phenotypes, loss of Brat resulted in reduced FM1-43 uptake at the NMJ terminals, indicating that brat regulates synaptic endocytosis. Genetic analysis revealed that the actions of Brat at synapses are mediated through mothers against decapentaplegic (Mad), the signal transduction effector of the bone morphogenetic protein (BMP) signaling pathway. Furthermore, biochemical analyses showed upregulated levels of Mad protein but normal mRNA levels in the larval brains of brat mutants, suggesting that Brat suppresses Mad translation. Consistently, knockdown of brat by RNA interference in Drosophila S2 cells also increased Mad protein level. These results together reveal an important and previously unidentified role for Brat in synaptic development and endocytosis mediated by suppression of BMP signaling.

A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis.

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.

Stiff matrix induces exosome secretion to promote tumour growth.

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

The architecture of kinesin-3 KLP-6 reveals a multilevel-lockdown mechanism for autoinhibition.

Autoinhibition of kinesin-3 ensures the proper spatiotemporal control of the motor activity for intracellular transport, but the underlying mechanism remains elusive. Here, we determine the full-length structure of kinesin-3 KLP-6 in a compact self-folded state. Unexpectedly, all the internal coiled-coil segments and domains in KLP-6 cooperate to successively lock down the neck and motor domains. The first coiled-coil segment is melted into several short helices that work with the motor domain to restrain the entire neck domain. The second coiled-coil segment associates with its neighboring FHA and MBS domains and integrates with the tail MATH domain to form a supramodule that synergistically wraps around the motor domain to trap the nucleotide and hinder the microtubule binding. This multilevel-lockdown mechanism for autoinhibition could be applicable to other kinesin-3 motors.

Characterization of a thermostable, protease-tolerant inhibitor of α-glycosidase from carrot: A potential oral additive for treatment of diabetes.

Inhibiting α-glucosidase activity is important in controlling postprandial hyperglycemia and, thus, helping to manage type-2 diabetes mellitus (T2DM). In the present study, we purified a hypothetical protein of carrots called DCHP (Daucus Carrot hypoglycemic peptide), and their inhibitory effects on α-glucosidase, as well as related mechanisms, were investigated. The recombinant DCHP protein with a molecular weight of 8 kDa showed strong inhibitory activity against α-glycosidase and maintained good stability in solution. DCHP exhibited no inhibitory activity but was tolerant to trypsin and chymotrypsin. Cellular experiments demonstrated that glucose consumption and lactic acid production increased rapidly when treated with DCHP in Caco-2 and HepG2 cells. DCHP crystal was generated, and the crystal structure, which was similar to that of rBTI and consisted of a central α-helix and a two-stranded ß-sheet with a unique loop region. The interaction between DCHP and α-glycosidase was investigated by molecular docking and site-directed mutation, which revealed that Glu43, Pro46, Thr47 Thr48 and Gln49 are the key residues in DCHP that inhibit α-glycosidase activity. This work provides potential bioactive peptides as functional foods or nutraceutical supplements in preventing and managing T2DM.

Intertwined Wdr47-NTD dimer recognizes a basic-helical motif in Camsap proteins for proper central-pair microtubule formation.

Calmodulin-regulated spectrin-associated proteins (Camsaps) bind to the N-terminal domain of WD40-repeat 47 (Wdr47-NTD; featured with a LisH-CTLH motif) to properly generate axonemal central-pair microtubules (CP-MTs) for the planar beat pattern of mammalian motile multicilia. The underlying molecular mechanism, however, remains unclear. Here, we determine the structures of apo-Wdr47-NTD and Wdr47-NTD in complex with a characteristic Wdr47-binding region (WBR) from Camsap3. Wdr47-NTD forms an intertwined dimer with a special cross-over region (COR) in addition to the canonical LisH and globular α-helical core (GAC). The basic WBR peptide adopts an α-helical conformation and anchors to a tailored acidic pocket embedded in the COR. Mutations in this target-binding pocket disrupt the interaction between Wdr47-NTD and Camsap3. Impairing Wdr47-Camsap interactions markedly reduces rescue effects of Wdr47 on CP-MTs and ciliary beat of Wdr47-deficient ependymal cells. Thus, Wdr47-NTD functions by recognizing a specific basic helical motif in Camsap proteins via its non-canonical COR, a target-binding site in LisH-CTLH-containing domains.

Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

Multi-site-mediated entwining of the linear WIR-motif around WIPI ß-propellers for autophagy.

WIPI proteins (WIPI1-4) are mammalian PROPPIN family phosphoinositide effectors essential for autophagosome biogenesis. In addition to phosphoinositides, WIPI proteins can recognize a linear WIPI-interacting-region (WIR)-motif, but the underlying mechanism is poorly understood. Here, we determine the structure of WIPI3 in complex with the WIR-peptide from ATG2A. Unexpectedly, the WIR-peptide entwines around the WIPI3 seven-bladed ß-propeller and binds to three sites in blades 1-3. The N-terminal part of the WIR-peptide forms a short strand that augments the periphery of blade 2, the middle segment anchors into an inter-blade hydrophobic pocket between blades 2-3, and the C-terminal aromatic tail wedges into another tailored pocket between blades 1-2. Mutations in three peptide-binding sites disrupt the interactions between WIPI3/4 and ATG2A and impair the ATG2A-mediated autophagic process. Thus, WIPI proteins recognize the WIR-motif by multi-sites in multi-blades and this multi-site-mediated peptide-recognition mechanism could be applicable to other PROPPIN proteins.

CASK modulates the assembly and function of the Mint1/Munc18-1 complex to regulate insulin secretion.

Calcium/calmodulin-dependent protein serine kinase (CASK) is a key player in vesicle transport and release in neurons. However, its precise role, particularly in nonneuronal systems, is incompletely understood. We report that CASK functions as an important regulator of insulin secretion. CASK depletion in mouse islets/ß cells substantially reduces insulin secretion and vesicle docking/fusion. CASK forms a ternary complex with Mint1 and Munc18-1, and this event is regulated by glucose stimulation in ß cells. The crystal structure of the CASK/Mint1 complex demonstrates that Mint1 exhibits a unique "whip"-like structure that wraps tightly around the CASK-CaMK domain, which contains dual hydrophobic interaction sites. When triggered by CASK binding, Mint1 modulates the assembly of the complex. Further investigation revealed that CASK-Mint1 binding is critical for ternary complex formation, thereby controlling Munc18-1 membrane localization and insulin secretion. Our work illustrates the distinctive molecular basis underlying CASK/Mint1/Munc18-1 complex formation and reveals the importance of the CASK-Mint1-Munc18 signaling axis in insulin secretion.

Structural Conservation of the Two Phosphoinositide-Binding Sites in WIPI Proteins.

WIPI proteins are mammalian PROPPIN family members that bind to phosphoinositides and play prominent roles in autophagosome biogenesis. Two phosphoinositide-binding sites were previously described in yeast PROPPIN Hsv2 but remain to be determined in mammalian WIPI proteins. Here, we characterized four human WIPI proteins (WIPI1-4) and solved the structure of WIPI3. WIPI proteins can bind to PI(3)P and PI(3,5)P2 and adopt a conventional seven-bladed ß-propeller fold. The structure of WIPI3 revealed that WIPI proteins also contain two sites embedded in blades 5 and 6 for recognizing phosphoinositides, resembling that in Hsv2. Structural comparison further demonstrated that the two conserved phosphoinositide-binding sites in PROPPIN proteins are not identical but intrinsically tend to recognize different types of phosphoinositides. This work provides the structural evidence to support the conservation of the two phosphoinositide-binding sites in WIPI proteins and also uncovers the potential phosphoinositide-binding selectivity for each site.

YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization.

As a master regulator of the macroautophagy/autophagy-lysosomal pathway, TFEB (transcription factor EB) plays a prominent role in regulating neurodegenerative diseases and cancer. The transcription activity of TFEB is tightly controlled by phosphorylation and dephosphorylation. Phosphorylated S211 (p-S211) of TFEB can be recognized by YWHA/14-3-3 proteins for TFEB cytoplasmic localization. Here, we characterized the interactions between phosphorylated TFEB and YWHA/14-3-3 proteins and determined the structures of YWHA/14-3-3 proteins in complex with a TFEB p-S211-peptide. Although the critical arginine for YWHA/14-3-3 recognition is missing in the N terminus of the TFEB p-S211-peptide, the C-terminal additional hydrophobic residues of the peptide unexpectedly occupy nearly half of the target-binding groove of YWHA/14-3-3 proteins, which compensates for the N-terminal defect and is distinct from the canonical YWHA/14-3-3-binding mode. Mutations of essential residues in the interaction interface between TFEB and YWHA/14-3-3 proteins disrupted their interactions and severely impaired the cytoplasmic localization of TFEB, which altered the expression of TFEB target genes and affected autophagy. Thus, YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization and its activity. Abbreviation: ACTB: actin beta; ALP: autophagy-lysosomal pathway; ATP6V1H: ATPase H+ transporting V1 subunit H; bHLH: basic helix-loop-helix; CLEAR: coordinated lysosomal expression and regulation; Co-IP: co-immunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MITF: melanocyte inducing transcription factor; NLS: nuclear localization signal; TFEB: transcription factor EB; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.