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Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm 2 ). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm 2 ). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression., Competing Interests: Competing interests statement:The authors declare no competing interest.

Zebra and quagga mussels ( Dreissena spp. ) are invasive freshwater biofoulers that perpetrate devastating economic and ecological impact. Their success depends on their ability to anchor onto substrates with protein-based fibers known as byssal threads. Yet, compared to other mussel lineages, little is understood about the proteins comprising their fibers or their evolutionary history. Here, we investigated the hierarchical protein structure of Dreissenid byssal threads and the process by which they are fabricated. Unique among bivalves, we found that threads possess a predominantly β -sheet crystalline structure reminiscent of spider silk. Further analysis revealed unexpectedly that the Dreissenid thread protein precursors are mechanoresponsive α -helical proteins that are mechanically processed into β -crystallites during thread formation. Proteomic analysis of the byssus secretory organ and byssus fibers revealed a family of ultrahigh molecular weight (354 to 467 kDa) asparagine-rich (19 to 20%) protein precursors predicted to form α -helical coiled coils. Moreover, several independent lines of evidence indicate that the ancestral predecessor of these proteins was likely acquired via horizontal gene transfer. This chance evolutionary event that transpired at least 12 Mya has endowed Dreissenids with a distinctive and effective fiber formation mechanism, contributing significantly to their success as invasive species and possibly, inspiring new materials design., Competing Interests: Competing interests statement:The authors declare no competing interest.

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin–ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

Nitrogenases utilize Fe-S clusters to reduce N 2 to NH 3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57 Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57 Fe labeling of the L-cluster-a carbide-containing, [Fe 8 S 9 C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57 Fe 2+ results in 57 Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57 Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors., Competing Interests: The authors declare no competing interest.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK + UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis., Competing Interests: The authors declare no competing interest.

Cerebellins are important neurexin ligands that remain incompletely understood. Two critical questions in particular remain unanswered: do different cerebellins perform distinct functions, and do these functions act in the initial establishment of synapses or in rendering nascent synapses capable of normal synaptic transmission? Here we show that in mice, Cbln2 and Cbln4 are expressed in the medial habenula (MHb) nucleus in different types of neurons that project to distinct target neurons in the interpeduncular nucleus. Conditional genetic deletion of Cbln2 in the MHb impaired synaptic transmission at Cbln2 + synapses in the interpeduncular neurons within 3 wk, but decreased synapse numbers only after 3 mo, suggesting a functional, but not a structural, requirement for Cbln2 in synapses formed by Cbln2-expressing neurons. In contrast, genetic deletions of Cbln4 in the MHb had no major effect on synaptic transmission or synapse numbers in interpeduncular target neurons. Nevertheless, MHb ablation of both Cbln2 and Cbln4 significantly impaired behavioral responses in mice, but affected different types of behaviors. Specifically, Cbln2 MHb deletions decreased spatial learning, as measured in the water T-maze, whereas Cbln4 MHb deletions increased anxiety levels, as monitored in the open field test and elevated plus maze. Thus, Cbln2 and Cbln4 are expressed in distinct MHb neurons that contribute to different behaviors., Competing Interests: The authors declare no conflict of interest.

Increased adrenomedullin (ADM) levels are associated with various cardiac diseases such as myocardial infarction (MI). ADM is cleaved off from the full-length precursor protein proadrenomedullin (ProADM) during its posttranslational processing. To date, no biological effect of ProADM is reported, while ADM infusion leads to antiapoptotic effects and improved cardiac function. Using an MI mouse model, we found an induction of ProADM gene as well as protein expression during the early phase of MI. This was accompanied by apoptosis and increasing inflammation, which substantially influence the post-MI remodeling processes. Simulating ischemia in vitro, we demonstrate that ProADM expression was increased in cardiomyocytes and cardiac fibroblasts. Subsequently, we treated ischemic cardiomyocytes with either ProADM or ADM and found that both proteins increased survival. This effect was diminishable by blocking the ADM 1 receptor. To investigate whether ProADM and ADM play a role in the regulation of cardiac inflammation, we analyzed chemokine expression after treatment of cells with both proteins. While ProADM induced an expression of proinflammatory cytokines, thus promoting inflammation, ADM reduced chemokine expression. On leukocytes, both proteins repressed chemokine expression, revealing antiinflammatory effects. However, ProADM but not ADM dampened concurrent activation of leukocytes. Our data show that the full-length precursor ProADM is biologically active by reducing apoptosis to a similar extent as ADM. We further assume that ProADM induces local inflammation in affected cardiac tissue but attenuates exaggerated inflammation, whereas ADM has low impact. Our data suggest that both proteins are beneficial during MI by influencing apoptosis and inflammation., Competing Interests: The authors declare no conflict of interest.

The δ1 glutamate receptor (GluD1) was cloned decades ago and is widely expressed in many regions of the brain. However, its functional roles in these brain circuits remain unclear. Here, we find that GluD1 is required for both excitatory synapse formation and maintenance in the hippocampus. The action of GluD1 is absent in the Cbln2 knockout mouse. Furthermore, the GluD1 actions require the presence of presynaptic neurexin 1β carrying the splice site 4 insert (+S4). Together, our findings demonstrate that hippocampal synapse assembly and maintenance require a tripartite molecular complex in which the ligand Cbln2 binds with presynaptic neurexin 1β (+S4) and postsynaptic GluD1. We provide evidence that this mechanism may apply to other forebrain synapses, where GluD1 is widely expressed., Competing Interests: The authors declare no conflict of interest.

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15 - and Cdhr23 -expressing interneuron precursors in Cdhr23 -/- and Cdhr15 -/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23 - and Cdhr15 -expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients., Competing Interests: The authors declare no conflict of interest.

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a β-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded β-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana , revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space., Competing Interests: The authors declare no conflict of interest.

The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.

Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentin(Y117L)). By tagging vimentin(Y117L) with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentin(Y117L) contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks.

The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

MicroRNAs (miRNAs) are small, 18-22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA-protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA-Gag complexes interfere with viral-RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA's target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.

Presecretory proteins are translocated across biological membranes through protein-conducting channels such as Sec61 (eukaryotes) and SecYEG (bacteria). SecA, a translocation ATPase, pushes preproteins out with dynamic structural changes through SecYEG. SecG, a subunit of the SecYEG channel possessing two transmembrane stretches (TMs), undergoes topology inversion coupled with SecA-dependent translocation. Recently, we characterized membrane protein integrase (MPIase), a glycolipozyme involved in not only protein integration into membranes but also preprotein translocation. We report here that SecG inversion occurs only when MPIase associates with SecYEG. We also found that MPIase modulates the dimer orientation of SecYEG. Cysteine-scanning mutagenesis mapped SecG TM 2 to a relatively hydrophilic environment. The dimer formation of SecG, crosslinked at TM 2, was not observed on SecG inversion, indicating that SecYEG undergoes a dynamic structural change during preprotein translocation.

Berninamycin is a member of the pyridine-containing thiopeptide class of antibiotics that undergoes massive posttranslational modifications from ribosomally generated preproteins. Berninamycin has a 2-oxazolyl-3-thiazolyl-pyridine core embedded in a 35-atom macrocycle rather than typical trithiazolylpyridine cores embedded in 26-atom and 29-atom peptide macrocycles. We describe the cloning of an 11-gene berninamycin cluster from Streptomyces bernensis UC 5144, its heterologous expression in Streptomyces lividans TK24 and Streptomyces venezuelae ATCC 10712, and detection of variant and incompletely processed scaffolds. Posttranslational maturation in S. lividans of both the wild-type berninamycin prepeptide (BerA) and also a T3A mutant generates macrocyclic compounds as well as linear variants, which have failed to form the pyridine and the macrocycle. Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework.

Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. As the first described plant peptide signal, systemin regulates antiherbivore defenses in the Solanaceae, but in other plant families, peptides with analogous activity have remained elusive. In the current study, we demonstrate that a member of the maize (Zea mays) plant elicitor peptide (Pep) family, ZmPep3, regulates responses against herbivores. Consistent with being a signal, expression of the ZmPROPEP3 precursor gene is rapidly induced by Spodoptera exigua oral secretions. At concentrations starting at 5 pmol per leaf, ZmPep3 stimulates production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivory defense. These include proteinase inhibitors and biosynthetic enzymes for production of volatile terpenes and benzoxazinoids. In accordance with gene expression data, plants treated with ZmPep3 emit volatiles similar to those from plants subjected to herbivory. ZmPep3-treated plants also exhibit induced accumulation of the benzoxazinoid phytoalexin 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside. Direct and indirect defenses induced by ZmPep3 contribute to resistance against S. exigua through significant reduction of larval growth and attraction of Cotesia marginiventris parasitoids. ZmPep3 activity is specific to Poaceous species; however, peptides derived from PROPEP orthologs identified in Solanaceous and Fabaceous plants also induce herbivory-associated volatiles in their respective species. These studies demonstrate that Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing functional homologs of systemin outside of the Solanaceae.

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

Formation of specific neuronal connections often involves competition between adjacent axons, leading to stabilization of the active terminal, while retraction of the less active ones. The underlying molecular mechanisms remain unknown. We show that activity-dependent conversion of pro-brain-derived neurotrophic factor (proBDNF) to mature (m)BDNF mediates synaptic competition. Stimulation of motoneurons triggers proteolytic conversion of proBDNF to mBDNF at nerve terminals. In Xenopus nerve-muscle cocultures, in which two motoneurons innervate one myocyte, proBDNF-p75(NTR) signaling promotes retraction of the less active terminal, whereas mBDNF-tyrosine-related kinase B (TrkB) p75NTR (p75 neurotrophin receptor) facilitates stabilization of the active one. Thus, proBDNF and mBDNF may serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, and activity-dependent conversion of proBDNF to mBDNF may regulate synapse elimination.

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.

Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is the most ancient member of the ASPP family of proteins and an evolutionarily conserved inhibitor of p53. iASPP is also a binding partner and negative regulator of p65RelA. Because p65RelA and the p53 family members often have opposite effects in controlling cell fate, it is important to understand the cellular context in which iASPP can regulate their activities. To address this question and to study the biological importance of iASPP in vivo, we generated a transgenic mouse in which iASPP expression is controlled by the Cre/loxP recombination system. We observed that iASPP is able to prevent premature cellular senescence in mouse embryonic fibroblasts. iASPP loss resulted in increased differentiation of primary keratinocytes both in vitro and in vivo. In stratified epithelia, nuclear iASPP often colocalized with p63 in the nuclei of basal keratinocytes. Consistent with this, iASPP bound p63 and inhibited the transcriptional activity of both TAp63α and ΔNp63α in vitro and influenced the expression level of p63-regulated genes such as loricrin and involucrin in vivo. In contrast, under the same conditions, p65RelA was frequently expressed as a cytoplasmic protein in the suprabasal layers of stratified epithelia and rarely colocalized with nuclear iASPP. Thus, iASPP is likely to control epithelial stratification by regulating p63's transcriptional activity, rather than p65RelA's. This study identifies iASPP as an inhibitor of senescence and a key player in controlling epithelial stratification.

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24(-/-) mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated aging-associated phenotypes, both in vitro and in vivo. The life span of Zmpste24(-/-) mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathy-based progeroid pathologies.

Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.

Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the protein-conducting channel SecYEG, and the motor protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to . To access the functional size of the SecYEG pore, the precursor of outer membrane protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane protein A conjugates carrying tetraarylmethanes with diameters up to , whereas a sized molecule blocked the translocation pore. Translocation of the protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of , the pore accommodates structures of at least , which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.

The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.

Leptin monotherapy reverses the deadly consequences and improves several of the metabolic imbalances caused by insulin-deficient type 1 diabetes (T1D) in rodents. However, the mechanism(s) underlying these effects is totally unknown. Here, we report that intracerebroventricular (icv) infusion of leptin reverses lethality and greatly improves hyperglycemia, hyperglucagonemia, hyperketonemia, and polyuria caused by insulin deficiency in mice. Notably, icv leptin administration leads to increased body weight while suppressing food intake, thus correcting the catabolic consequences of T1D. Also, icv leptin delivery improves expression of the metabolically relevant hypothalamic neuropeptides proopiomelanocortin, neuropeptide Y, and agouti-related peptide in T1D mice. Furthermore, this treatment normalizes phosphoenolpyruvate carboxykinase 1 contents without affecting glycogen levels in the liver. Pancreatic β-cell regeneration does not underlie these beneficial effects of leptin, because circulating insulin levels were undetectable at basal levels and following a glucose overload. Also, pancreatic preproinsulin mRNA was completely absent in these icv leptin-treated T1D mice. Furthermore, the antidiabetic effects of icv leptin administration rapidly vanished (i.e., within 48 h) after leptin treatment was interrupted. Collectively, these results unveil a key role for the brain in mediating the antidiabetic actions of leptin in the context of T1D.

Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and S-adenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75(NTR). The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75(NTR)-regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-alpha converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.

Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a "clamp" of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel.

We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin alpha suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.

Gram-positive bacteria elaborate pili and do so without the participation of folding chaperones or disulfide bond catalysts. Sortases, enzymes that cut pilin precursors, form covalent bonds that link pilin subunits and assemble pili on the bacterial surface. We determined the x-ray structure of BcpA, the major pilin subunit of Bacillus cereus. The BcpA precursor encompasses 2 Ig folds (CNA(2) and CNA(3)) and one jelly-roll domain (XNA) each of which synthesizes a single intramolecular amide bond. A fourth amide bond, derived from the Ig fold of CNA(1), is formed only after pilin subunits have been incorporated into pili. We report that the domains of pilin precursors have evolved to synthesize a discrete sequence of intramolecular amide bonds, thereby conferring structural stability and protease resistance to pili.

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR(2)) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR(2) or the use of PAR(2)(-/-) mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its alpha(2)- and beta-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.

Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metalloproteases. The Bacillus subtilis SpoIVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-sigma(K), releasing sigma(K) to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpoIVFB, expression in E. coli was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-sigma(K). Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-beta-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-sigma(K) in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-sigma(K) and ATP changed the interaction, suggesting that ATP regulates substrate access to the active site and renders cleavage sensitive to the cellular energy level, which may be a general feature of CBS-domain-containing IPs. Incorporation of SpoIVFB into preformed liposomes stimulated its ability to cleave Pro-sigma(K). Cleavage depended on ATP and the correct peptide bond was shown to be cleaved using a rapid and sensitive mass spectrometry assay. A system for biochemical studies of RIP by a metalloprotease in a membrane environment has been established using methods that might be applicable to other IPs.

Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane proteins called tyrosylprotein sulfotransferases (TPSTs). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis MM2d cells, and identified a 62-kDa protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. This protein is a 500-aa type I transmembrane protein that shows no sequence similarity to animal TPSTs. A recombinant version of this protein expressed in yeast catalyzed tyrosine sulfation of both PSY1 and PSK precursor polypeptide in vitro, indicating that the newly identified protein is indeed an Arabidopsis (At)TPST. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence, and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal-optimal or optimal-optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI-III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.

Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we show that low-frequency stimulation induced predominant proBDNF secretion in cultured hippocampal neurons. In contrast, high-frequency stimulation preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular cleavage of proBDNF greatly reduced high-frequency stimulation-induced extracellular mBDNF. Moreover, high-frequency, but not low-frequency stimulation selectively induced the secretion of tissue plasminogen activator, a key protease involved in extracellular proBDNF to mBDNF conversion. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF/mBDNF by regulating the secretion of extracellular proteases. Our study demonstrates activity-dependent control of extracellular proteolytic cleavage of a secretory protein, and reveals an important mechanism that controls diametrically opposed functions of BDNF isoforms.

Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.

Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the alpha-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased alpha-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.

We identified an antigen recognized on a human non-small-cell lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide is presented by HLA-A2 and is encoded by the CALCA gene, which codes for calcitonin and for the alpha-calcitonin gene-related peptide. The peptide is derived from the carboxy-terminal region of the preprocalcitonin signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing. Processing occurs within the endoplasmic reticulum of all tumoral and normal cells tested, including dendritic cells, and it involves signal peptidase and the aspartic protease, signal peptide peptidase. The CALCA gene is overexpressed in medullary thyroid carcinomas and in several lung carcinomas compared with normal tissues, leading to recognition by the T cell clone. This new epitope is, therefore, a promising candidate for cancer immunotherapy.

Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin gene-related peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-beta-endorphin (ppbetaEP) led to significant (P < 0.0001) reversal of mechanical allodynia for >/=3 months. The antiallodynic effect could be reversed by the mu-opioid antagonist naloxone 4 months after gene transfer (P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with ppbetaEP was equally effective (P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys --> Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with beta cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially beta cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of beta cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

HIV protease inhibitors (HIV-PIs) target the HIV aspartyl protease, which cleaves the HIV gag-pol polyprotein into shorter proteins required for the production of new virions. HIV-PIs are a cornerstone of treatment for HIV but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we show that HIV-PIs caused an accumulation of prelamin A. The prelamin A in HIV-PI-treated fibroblasts migrated more rapidly than nonfarnesylated prelamin A, comigrating with the farnesylated form of prelamin A that accumulates in ZMPSTE24-deficient fibroblasts. The accumulation of farnesyl-prelamin A in response to HIV-PI treatment was exaggerated in fibroblasts heterozygous for Zmpste24 deficiency. HIV-PIs inhibited the endoproteolytic processing of a GFP-prelamin A fusion protein. The HIV-PIs did not affect the farnesylation of HDJ-2, nor did they inhibit protein farnesyltransferase in vitro. HIV-PIs also did not inhibit the activities of the isoprenyl-cysteine carboxyl methyltransferase ICMT or the prenylprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC(50): lopinavir, 18.4 +/- 4.6 microM; tipranavir, 1.2 +/- 0.4 microM). We conclude that the HIV-PIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. The inhibition of ZMPSTE24 by HIV-PIs could play a role in the side effects of these drugs.

Th1 and Th2 cells represent the two main functional subsets of CD4(+) T helper cell, and are defined by their cytokine expression. Human Th1 cells express IFNgamma, whilst Th2 cells express IL-4, IL-5, and IL-13. Th1 and Th2 cells have distinct immunological functions, and can drive different immunopathologies. Here, we show that in vitro-differentiated human Th2 cells highly selectively express the gene for pro-melanin-concentrating hormone (PMCH), using real-time RT-PCR, enzyme immunoassay, and Western blot analysis. PMCH encodes the prohormone, promelanin-concentrating hormone (PMCH), which is proteolytically processed to produce several peptides, including the orexigenic hormone melanin-concentrating hormone (MCH). PMCH expression by Th2 cells was activation responsive and increased throughout the 28-day differentiation in parallel with the expression of the Th2 cytokine genes. MCH immunoreactivity was detected in the differentiated Th2 but not Th1 cell culture supernatants after activation, and contained the entire PMCH protein, in addition to several smaller peptides. Human Th1 and Th2 cells were isolated by their expression of IFNgamma and CRTH2, respectively, and the ex vivo Th2 cells expressed PMCH upon activation, in contrast to the Th1 cells. Because Th2 cells are central to the pathogenesis of allergic diseases including asthma, expression of PMCH by activated Th2 cells in vivo may directly link allergic inflammation to energy homeostasis and may contribute to the association between asthma and obesity.

Plastids are semiautonomous organelles that contain only limited coding information in their own DNA. Because most of their genome was transferred to the nucleus after their endosymbiotic origin, plastids must import the major part of their protein constituents from the cytosol. The exact role of cytosolic targeting factors in the regulation of plastid protein import has not been determined. Here, we report that the nucleus-encoded NADPH:protochlorophyllide (Pchlide) oxidoreductase A plastid precursor (pPORA) can use two different plastid import pathways that differ by the requirements for cytosolic 14:3:3 proteins and Hsp70. pPORA synthesized in a wheat germ lysate segregated into different precursor fractions. While import of free pPORA and only Hsp70-complexed pPORA was Pchlide-dependent and involved the previously identified Pchlide-dependent translocon, 14:3:3 protein- and Hsp70-complexed pPORA was transported into Pchlide-free chloroplasts through the Toc75-containing standard translocon at the outer chloroplast membrane/translocon at the inner chloroplast membrane machinery. A 14:3:3 protein binding site was identified in the mature region of the (35)S-pPORA, which governed 14:3:3 protein- and Hsp70-mediated, Pchlide-independent plastid import. Collectively, our results reveal that the import of pPORA into the plastids is tightly regulated and involves different cytosolic targeting factors and plastid envelope translocon complexes.

The high-resolution crystal structure of kexin (Kex2) in complex with a peptidyl-chloromethylketone inhibitor containing a noncognate lysine at the P(1) position provides the structural basis for the differential lysine/arginine selectivity that defines the prohormone (proprotein) convertase (PC) family. By comparison with the previous structures of Kex2 and furin, this structure of the acylated enzyme provides a basis for the observed decrease in the acylation rate with substrates containing a lysine at P(1) and the absence of an effect on the deacylation rate without involving mobility of the S(1) lid. The structure of the complex shows that a secondary subsite in the S(1) pocket is present, and that this site recognizes and binds the P(1) lysine in a more shallow fashion than arginine. This results in a displacement of the bound peptide away from the S385 nucleophile relative to substrates containing a P(1) arginine. It is concluded that this alternate binding site and resultant displacement of the scissile bond in the active site results in the observed decrease in the acylation rate. Studies of the inactivation kinetics of Kex2 by two peptidyl chloromethylketone inhibitors demonstrates that the selectivity between lysine and arginine at the P(1) position arises at the acylation step, consistent with what was observed with peptidyl substrates [Rockwell NC, Fuller RS (2001) J Biol Chem 276:38394-38399].

Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75NTR) and sortilin. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus). Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. Here we describe the construction, expression, and purification of this more stable proNGF molecule. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Expression was performed in stably transfected Sf21 insect cells. Purification involved strong cation-exchange chromatography and N60 immunoaffinity column chromatography. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells.

The aberrant production of precursor IL-1alpha (pre-IL-1alpha) in skin fibroblasts that are derived from systemic sclerosis (SSc) is associated with the induction of IL-6 and procollagen, which contributes to the fibrosis of SSc. However, little is understood about how intracellular pre-IL-1alpha regulates the expression of the other molecules in fibroblasts. We report here that pre-IL-1alpha can form a complex with IL-1alpha-binding proteins that is translocated into the nuclei of fibroblasts. Immunoprecipitation that used anti-human IL-1alpha Ab and (35)S-labeled nuclear extracts of fibroblasts showed three specific bands (approximately equal to 31, 35, and 65 kDa). The 31-kDa molecule was identified as pre-IL-1alpha, and the 35- and 65-kDa molecules might be pre-IL-1alpha-binding proteins. A partial sequencing for the 10 aa from the N-terminals of the molecules showed 100% homology for HAX-1 (HS1-associated protein X-1) and IL-1 receptor type II (IL-1RII). Suppression of the genes of HAX-1 or IL-1RII induced the inhibitory effects of IL-1 signal transduction, including production of IL-6 and procollagen, by fibroblasts. In particular, pre-IL-1alpha was not translocated into the nucleus by an inhibition of HAX-1. These findings reveal that nuclear localization of pre-IL-1alpha depends on the binding to HAX-1 and that biological activities might be elicited by the binding to both HAX-1 and IL-1RII in SSc fibroblasts.

Innate immunity is initiated in animals and plants through the recognition of a variety of pathogen-associated molecules that in animals are called pathogen-associated molecular patterns and in plants are called elicitors. Some plant pathogen-derived elicitors have been identified as peptides, but peptide elicitors derived from the plant itself that activate defensive genes against pathogens have not been previously identified. Here, we report the isolation and characterization of a 23-aa peptide from Arabidopsis, called AtPep1, which activates transcription of the defensive gene defensin (PDF1.2) and activates the synthesis of H(2)O(2), both being components of the innate immune response. The peptide is derived from a 92-aa precursor encoded within a small gene that is inducible by wounding, methyl jasmonate, and ethylene. Constitutive expression of the AtPep1 precursor gene PROPEP1 in transgenic Arabidopsis plants causes a constitutive transcription of PDF1.2. When grown in soil, the transgenic plants exhibited an increased root development compared with WT plants and an enhanced resistance toward the root pathogen Pythium irregulare. Six paralogs of PROPEP1 are present in Arabidopsis, and orthologs have been identified in species of several agriculturally important plant families, where they are of interest for their possible use in crop improvement.