Short N-terminal Fragment Research Articles

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies.

The RAB3-RIM Pathway Is Essential for the Release of Neuromodulators

Secretion principles are conserved from yeast to humans, and many yeast orthologs have established roles in synaptic vesicle exocytosis in the mammalian brain. Surprisingly, SEC4 orthologs and their effectors, the exocyst, are dispensable for synaptic vesicle exocytosis. Here, we identify the SEC4 ortholog RAB3 and its neuronal effector, RIM1, as essential molecules for neuropeptide and neurotrophin release from dense-core vesicles (DCVs) in mammalian neurons. Inactivation of all four RAB3 genes nearly ablated DCV exocytosis, and re-expression of RAB3A restored this deficit. In RIM1/2-deficient neurons, DCV exocytosis was undetectable. Full-length RIM1, but not mutants that lack RAB3 or MUNC13 binding, restored release. Strikingly, a short N-terminal RIM1 fragment only harboring RAB3- and MUNC13-interacting domains was sufficient to support DCV exocytosis. We propose that RIM and MUNC13 emerged as mammalian alternatives to the yeast exocyst complex as essential RAB3/SEC4 effectors and organizers of DCV fusion sites by recruiting DCVs via RAB3.

A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions

Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody-epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding

Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Single Molecule Visualization of Cardiac Myosin-Binding Protein C N-Terminal Fragments Interacting with Regulated Actin Filaments: Mechanisms of Calcium Sensitization

Tropomyosin movement on actin resulting from calcium binding to troponin permits myosin binding, filament sliding and force generation in the cardiac muscle sarcomere. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism is that cMyBP-C binds directly to the regulated actin filament at low calcium levels to move tropomyosin, thus recruiting myosin binding. To test this, we directly visualized fluorescently-labelled cMyBP-C N-terminal fragments and GFP-labelled myosin molecules binding to regulated actin filaments in a single molecule assay. Binding of cMyBP-C's C0C3 N-terminal domains to regulated actin enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 fragments bound cooperatively, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that utilizes a weak-binding mode to search for access to actin and a tight-binding mode that enhances myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to regulated actin filaments similarly to the Cy3-C0C3 fragment, the shorter fragments were unable to affect myosin binding. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Cardioprotective properties of N-terminal galanin fragment (2-15) in experimental ischemia/reperfusion injury.

Background and purposeGalanin is an endogenous peptide involved in diverse physiological functions in the central nervous system including central cardiovascular regulation. The present study was designed to evaluate the potential effects of the short N-terminal galanin fragment 2-15 (G) on cardiac ischemia/reperfusion (I/R) injury.Experimental ApproachPeptide G was synthesized by the automatic solid phase method and identified by 1H-NMR spectroscopy and mass spectrometry. Experiments were performed on cultured rat cardiomyoblast (H9C2) cells, isolated perfused working rat hearts and anaesthetized open-chest rats.Key ResultsCell viability increased significantly after treatment with 10 and 50 nM of G peptide. In hypoxia and reoxygenation conditions, exposure of H9C2 cells to G peptide decreased cell apoptosis and mitochondrial reactive oxygen species (ROS) production. Postischemic infusion of G peptide reduced cell membrane damage and improved functional recovery in isolated hearts during reperfusion. These effects were accompanied by enhanced restoration of myocardial metabolic state. Treatment with G peptide at the onset of reperfusion induced minor changes in hemodynamic variables but significantly reduced infarct size and plasma levels of necrosis markers.Conclusion and implicationsThese findings suggest that G peptide is effective in mitigating cardiac I/R injury, thereby providing a rationale for promising tool for the treatment of cardiovascular diseases.

The A2V mutation as a new tool for hindering A\u03b2 aggregation: A neutron and x-ray diffraction study

We have described a novel C-to-T mutation in the APP gene that corresponds to an alanine to valine substitution at position 673 in APP (A673V), or position 2 of the amyloid-β (Aβ) sequence. This mutation is associated with the early onset of AD-type dementia in homozygous individuals, whereas it has a protective effect in the heterozygous state. Correspondingly, we observed differences in the aggregation properties of the wild-type and mutated Aβ peptides and their mixture. We have carried out neutron diffraction (ND) and x-ray diffraction (XRD) experiments on magnetically-oriented fibers of Aβ1-28WT and its variant Aβ1-28A2V. The orientation propensity was higher for Aβ1-28A2V suggesting that it promotes the formation of fibrillar assemblies. The diffraction patterns by Aβ1-28WT and Aβ1-28A2V assemblies differed in shape and position of the equatorial reflections, suggesting that the two peptides adopt distinct lateral packing of the diffracting units. The diffraction patterns from a mixture of the two peptides differed from those of the single components, indicating the presence of structural interference during assembly and orientation. The lowest orientation propensity was observed for a mixture of Aβ1-28WT and a short N-terminal fragment, Aβ1-6A2V, which supports a role of Aβ’s N-terminal domain in amyloid fibril formation.

DsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication.

Enterovirus 71 (EV-A71) causes hand, foot and mouth disease in young children and infants, but can also cause severe neurological complications or even death. The double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), an interferon-induced antiviral protein, phosphorylates the regulatory α-subunit of the eukaryotic translation initiation factor 2 in response to viral infection, thereby blocking the translation of cellular and viral mRNA and promoting apoptosis. The cleavage of PKR after infection with poliovirus, a prototype enterovirus, has been reported by others, but the underlying mechanism of this cleavage and its role in viral replication remain unclear. In the present study, we show that viral 3C protease cleaves PKR at a site, Q188, which differs from the site cleaved during apoptosis, D251. In contrast to the conventional phosphorylation of PKR by dsRNA, EV-A71 3C physically interacts with PKR to mediate the phosphorylation of PKR; this effect is dependent on 3C protease activity. Overexpression of a catalytically inactive PKR mutant (K296H) accelerates viral protein accumulation and increases virus titer, whereas a K64E substitution in the dsRNA binding site abolishes this advantage. We also demonstrate that PKR cleavage mediated by EV-A71 3C protease produces a short N-terminal PKR fragment that can enhance EV-A71 replication, in terms of viral RNA, viral protein, and viral titers. We conclude that PKR is co-opted by EV-A71 via viral protease 3C-mediated proteolytic activation to facilitate viral replication.

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20–30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Short N-terminal galanin fragments are occurring naturally in vivo

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL1, GAL2, and GAL3, binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with 125I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced 125I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species. Biological significanceThis study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.

N-terminal chemical protein labeling using the naturally split GOS-TerL intein.

Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Chronological Profiling of Plasma Native Peptides after Hepatectomy in Pigs: Toward the Discovery of Human Biomarkers for Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a time-dependent process, which is tightly regulated by multiple signaling cascades. Failure of this complex process leads to posthepatectomy liver failure (PHLF), which is associated with a high rate of mortality. Thus, it is extremely important to establish a useful biomarker of liver regeneration to help prevent PHLF. Here, we hypothesized that alterations in the plasma peptide profile may predict liver regeneration following PHx and hence we set up a diagnostic platform for monitoring posthepatectomy outcome. We chronologically analyzed plasma peptidomic profiles of 5 partially hepatectomized microminipigs using the ClinProtTM system, which consists of magnetic beads and MALDI-TOF/TOF MS. We identified endogenous circulating peptides specific to each phase of the postoperative course after PHx in pigs. Notably, peptide fragments of histones were detected immediately after PHx; the presence of these fragments may trigger liver regeneration in the very acute phase after PHx. An N-terminal fragment of hemoglobin subunit α (3627 m/z) was detected as an acute-phase-specific peptide. In the recovery phase, the short N-terminal fragments of albumin (3028, 3042 m/z) were decreased, whereas the long N-terminal fragment of the protein (8926 m/z) was increased. To further validate and extract phase-specific biomarkers using plasma peptidome after PHx, plasma specimens of 4 patients who underwent PHx were analyzed using the same method as we applied to pigs. It revealed that there was also phase-specificity in peptide profiles, one of which was represented by a fragment of complement C4b (2378 m/z). The strategy described herein is highly efficient for the identification and characterization of peptide biomarkers of liver regeneration in a swine PHx model. This strategy is feasible for application to human biomarker studies and will yield clues for understanding liver regeneration in human clinical trials.

A Short N-Terminal Fragment in Front of the A1 Domain of Von Willebrand Factor Prevents the A1 from Binding the Platelet By Masking a Region Clustered By Type 2B VWD Mutations

Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb.We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region.Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing.

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

A Short Amino-Terminal Part of Arabidopsis Phytochrome A Induces Constitutive Photomorphogenic Response

Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana. phyA accumulates at high levels in the cytoplasm of etiolated seedlings, and light-induced phyA signaling is mediated by a complex regulatory network. This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo. It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro. To test the biological activity of this N-terminal fragment of phyA in planta, we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD, PHYA406-YFP-DD-NLS (nuclear localization signal), and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins. Here, we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns. Our results show that these truncated phyA proteins are light-stable, they trigger a constitutive photomorphogenic-like response when localized in the nuclei, and neither of them induces proper phyA signaling. We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1, a general repressor of photomorphogenesis, and co-localize with it in nuclear bodies. Thus, we conclude that, in planta, the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion.

Identification of an Ubinuclein 1 Region Required for Stability and Function of the Human HIRA/UBN1/CABIN1/ASF1a Histone H3.3 Chaperone Complex

The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin and is linked to gene activation, repression, and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1-175 is necessary and sufficient for interaction with the WD repeats of HIRA and attributed this interaction to a region from residues 120-175 that is highly conserved with the yeast ortholog Hpc2 and so termed the HRD for Hpc2-related domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41-77 of UBN1, which we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo, and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.

Capucin does not modify the toxicity of a mutant Huntingtin fragment in vivo

Genes selectively expressed in the striatum may be involved in the preferential vulnerability of striatal neurons to Huntington's disease (HD). Here, we investigated whether perturbations of Capucin expression, which is enriched in the striatum and downregulated in Huntington's disease models, could modify the neurotoxicity induced by the injection of a lentiviral vector encoding a short N-terminal fragment of mutant Huntingtin (mHtt) into the mouse striatum. Neither constitutive Capucin deficiency in knockout mice nor lentiviral vector-mediated Capucin overexpression in the striatum of adult wild type mice significantly modified vulnerability to the mHtt fragment in vivo, suggesting that Capucin has no impact on mHtt toxicity.

Probing Intein-Catalyzed Thioester Formation by Unnatural Amino Acid Substitutions in the Active Site

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers β-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.

The Nucleoporin Nup358/RanBP2 Promotes Nuclear Import in a Cargo‐ and Transport Receptor‐Specific Manner

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/β-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.

Ran-dependent docking of importin-β to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.