The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for ...analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.
At the center of the nuclear pore complex (NPC) is a uniquely versatile central transport channel. Structural analyses of distinct segments (“protomers”) of the three “channel” nucleoporins yielded a ...model for how this channel is constructed. Its principal feature is a midplane ring that can undergo regulated diameter changes of as much as an estimated 30 nm. To better understand how a family of “adaptor” nucleoporins—concentrically surrounding this channel—might cushion these huge structural changes, we determined the crystal structure of one adaptor nucleoporin, Nup157. Here, we show that a recombinant Saccharomyces cerevisiae Nup157 protomer, representing two-thirds of Nup157 (residues 70–893), folds into a seven-bladed β-propeller followed by an α-helical domain, which together form a C-shaped architecture. Notably, the structure contains a large patch of positively charged residues, most of which are evolutionarily conserved. Consistent with this surface feature, we found that Nup157 ₇₀–₈₉₃ binds to nucleic acids, although in a sequence-independent manner. Nevertheless, this interaction supports a previously reported role of Nup157, and its paralogue Nup170, in chromatin organization. Based on its nucleic acid binding capacity, we propose a dual location and function of Nup157. Finally, modeling the remaining C-terminal portion of Nup157 shows that it projects as a superhelical stack from the compact C-shaped portion of the molecule. The predicted four hinge regions indicate an intrinsic flexibility of Nup157, which could contribute to structural plasticity within the NPC.
During microbial infection, bystander CD8+ T cells that are not specific to infecting pathogens can be activated by interleukin (IL)-15. However, the tissue-homing properties of bystander-activated ...CD8+ T cells have not been elucidated. Here, we examine the effects of IL-15 on the expression of chemokine receptors on CD8+ T cells and their migration. IL-15 upregulates CCR5 in memory CD8+ T cells in the absence of T cell receptor (TCR) stimulation and enhances CCR5-dependent migration. IL-15-induced CCR5 upregulation is abrogated by TCR stimulation, indicating that CCR5 is upregulated in bystander-activated CD8+ T cells. Moreover, CCR5 signals increase proliferation and cytotoxic protein expression in IL-15-treated memory CD8+ T cells, although the increase has a small extent. CCR5 upregulation in bystander-activated CD8+ T cells is associated with severe liver injury in patients with acute hepatitis A. Altogether, the results indicate that CCR5 upregulation by IL-15 mediates the migration of bystander-activated CD8+ T cells.
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•IL-15 upregulates CCR5 in memory CD8+ T cells in the absence of TCR stimulation•CCR5 is upregulated in IL-15-induced bystander-activated CD8+ T cells•IL-15-treated CD8+ T cells migrate in a CCR5-dependent manner•CCR5 upregulation is associated with liver injury during acute hepatitis A
IL-15 induces TCR-independent bystander activation of memory CD8+ T cells. Seo et al. demonstrate that IL-15 upregulates CCR5 expression on memory CD8+ T cells and enhances CCR5-mediated migration. During acute hepatitis A, CCR5 is upregulated in bystander-activated CD8+ T cells and associated with liver injury.
The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a β propeller and an ...α-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.
NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other ...hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.
p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression ...of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.
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•Cancer cells can subvert wild-type p53 by expression of HDM2 and HDMX•HDM2 and dual inhibitors are in trials but selective HDMX agents are lacking•Scanning mutagenesis of a stapled p53 peptide identified HDMX-selective constructs•Structures of the complexes revealed a mechanism for HDMX-binding selectivity
Ben-Nun et al. performed scanning mutagenesis of a stapled-peptide dual inhibitor of HDM2/HDMX to identify mutations that conferred HDMX selectivity. Structures of L26E-mutant constructs in complex with HDMX revealed the molecular basis for binding selectivity. Stapled-peptide libraries can provide key structure-function insights, such as this blueprint for developing selective HDMX inhibitors.
We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three ...crystal structures of the nucleoporin pair Seh1•Nup85, which is part of the coat cylinder. The Seh1•Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13•Nup145C. Furthermore, the Seh1•Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1•Nup85 and Sec13•Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.
Abstract
Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B
N642H
, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell ...leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B
N642H
in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B
N642H
-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B
N642H
patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B
N642H
crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B
N642H
can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B
N642H
, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5B
N642H
activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.
Abstract
The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy ...against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.
Enteric fever is a systemic disease caused by Salmonella enterica serovar Typhi or Salmonella enterica serovar Paratyphi, characterized by high fever and abdominal pain. Most patients with enteric ...fever improve within a few days after antibiotic treatment. However, some patients do not recover as easily and develop fatal life-threatening complications, including intestinal hemorrhage. Lower gastrointestinal bleeding has been reported in 10% of cases. However, upper gastrointestinal bleeding has rarely been reported in patients with enteric fever. We present a case of gastric ulcer hemorrhage caused by enteric fever.
A 32-year-old woman, complaining of fever lasting four days and right upper quadrant pain and melena that started one day before admission, consulted our hospital. Abdominal computed tomography revealed mild hepatomegaly and gastroscopy revealed multiple active gastric ulcers with flat black hemorrhagic spots. The melena of the patient stopped on the third day. On the fifth admission day, she developed hematochezia. At that time, Salmonella enterica serovar Typhi was isolated from the blood culture. The antibiotic regimen was switched to ceftriaxone. Her hematochezia spontaneously resolved the following day. Finally, the patient was discharged on the 12th admission day without clinical symptoms. However, her fever recurred one month after discharge, and she was readmitted and Salmonella enterica serovar Typhi was confirmed again via blood culture. She was treated with ceftriaxone for one month, and was discharged without complications.
Our case showed that although rare, active gastric ulcers can develop in patients with enteric fever. Therefore, upper and lower gastrointestinal bleeding should be suspected in patients with enteric fever, especially showing relapsing bacteremia.
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