Three receptors (Rpn1/S2/PSMD2, Rpn10/S5a, Rpn13/Adrm1) in the proteasome bind substrates by interacting with conjugated ubiquitin chains and/or shuttle factors (Rad23/HR23, Dsk2/PLIC/ubiquilin, ...Ddi1) that carry ubiquitinated substrates to proteasomes. We solved the structure of two such receptors with their preferred shuttle factor, namely hRpn13Pru:hPLIC2UBL and scRpn1 T1:scRad23UBL. We find that ubiquitin folds in Rad23 and Dsk2 are fine-tuned by residue substitutions to achieve high affinity for Rpn1 and Rpn13, respectively. A single substitution in hPLIC2 yields enhanced interactions with the Rpn13 ubiquitin contact surface and sterically blocks hRpn13 binding to its preferred ubiquitin chain type, K48-linked chains. Rpn1 T1 binds two ubiquitins in tandem and we find that Rad23 binds exclusively to the higher-affinity Helix28/Helix30 site. Rad23 contacts at Helix28/Helix30 are optimized compared to ubiquitin by multiple conservative amino acid substitutions. Thus, shuttle factors deliver substrates to proteasomes through fine-tuned ubiquitin-like surfaces.
Display omitted
•UBL shuttle factors bind to ubiquitin-interacting residues of proteasome receptors•hRpn13:hPLIC2 structure reveals an optimized UBL for hRpn13 binding•hPLIC2 sterically blocks Rpn13 binding to K48 diubiquitin, its preferred chain type•Rpn1 T1:Rad23UBL structure reveals Rad23 binding to Helix28/Helix30 of Rpn1 T1
Chen et al. solve the hRpn13Pru:hPLIC2UBL and Rpn1 T1:Rad23UBL structures, which reveal how proteasome substrate receptors are targeted by substrate-carrying shuttle factors. Within each shuttle factor, a common ubiquitin-like surface is fine-tuned by conservative amino acid substitutions to bind a specific receptor.
Inflammatory gastrointestinal (GI) diseases and malignancies are associated with growing morbidity and cancer-related mortality worldwide. GI tumor and inflammatory cells contain activated ...sphingolipid-metabolizing enzymes, including sphingosine kinase 1 (SphK1) and SphK2, that generate sphingosine-1-phosphate (S1P), a highly bioactive compound. Many inflammatory responses, including lymphocyte trafficking, are directed by circulatory S1P, present in high concentrations in both the plasma and the lymph of cancer patients. High fat and sugar diet, disbalanced intestinal flora, and obesity have recently been linked to activation of inflammation and SphK/S1P/S1P receptor (S1PR) signaling in various GI pathologies, including cancer. SphK1 overexpression and activation facilitate and enhance the development and progression of esophageal, gastric, and colon cancers. SphK/S1P axis, a mediator of inflammation in the tumor microenvironment, has recently been defined as a target for the treatment of GI disease states, including inflammatory bowel disease and colitis. Several SphK1 inhibitors and S1PR antagonists have been developed as novel anti-inflammatory and anticancer agents. In this review, we analyze the mechanisms of SphK/S1P signaling in GI tissues and critically appraise recent studies on the role of SphK/S1P/S1PR in inflammatory GI disorders and cancers. The potential role of SphK/S1PR inhibitors in the prevention and treatment of inflammation-mediated GI diseases, including GI cancer, is also evaluated.
Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ...ubiquitin recognition and identified a sixth receptor, Rpn1. A site ( T1: ) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like ( UBL: ) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site ( T2: ) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
GTP-Dependent K-Ras Dimerization Muratcioglu, Serena; Chavan, Tanmay S.; Freed, Benjamin C. ...
Structure,
07/2015, Letnik:
23, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been ...suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.
Display omitted
•The GTP-bound K-Ras4B catalytic domain can form stable homodimers•The K-Ras4B predicted β-sheet dimer interface overlaps the effectors' binding site•The K-Ras4B α3/α4 helical dimer interface may promote Raf dimerization•The two interfaces may illuminate K-Ras4B nanocluster formation
Muratcioglu et al. show that GTP-bound but not GDP-bound K-Ras4B catalytic domain can form stable homodimers. Modeling reveals two major dimer interfaces. The highly populated β-sheet interface is at the Switch I/effector binding regions. The second major dimerization mode involves helical interfaces at the C-terminal allosteric lobe of the G domain.
Nanotechnology for Alzheimer Disease Leszek, Jerzy; Md Ashraf, Ghulam; Tse, Wai Hei ...
Current Alzheimer research,
01/2017, Letnik:
14, Številka:
11
Journal Article
Recenzirano
Alzheimer disease (AD) typically affects behavior, memory and thinking. The change in brain have been reported to begin approx. 10-20 years before the appearance of actual symptoms and diagnosis of ...AD. An early stage diagnosis and treatment of this lethal disease is the prime challenge, which is mainly halted by the lack of validated biomarkers.
Recent nanotechnological advancements have the potential to offer large scale effective diagnostic and therapeutic options. Targeted drug (e.g. Rivastigmine) delivery with the help of nanoparticles (NPs) in the range of 1-100 nm diameters can effectively cross the blood brain barrier with minimized side effects. Moreover, biocompatible nanomaterials with increased magnetic and optical properties can act as excellent alternative agents for an early diagnosis. With the high volume of research coming in support of the effective usage of NP based drug delivery in critical environment of CNS, it is quite likely that this approach can end up providing remarkable breakthroughs in early stage diagnosis and therapy of AD.
In the current review, we have presented a comprehensive outlook on the current challenges in diagnosis and therapy of AD, with an emphasis on the effective options provided by biocompatible NPs as imaging contrast agents and drug carriers.
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal ...transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 Å resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.
Structural and mechanical characterization of electron beam additive manufactured stainless steel samples has been carried out. The XRD measured austenite and ferrite lattice parameters showed their ...sensitivity to the heat input value, which was related to the chromium atom redistribution. The ferrite content depended on the heat input too. Optimal heat input level has been detected, which allowed obtaining the tensile strength higher than that of the base stainless steel. Residual strain levels in the as-deposited metal and fusion line zone have been measured using the X-ray sin
2
ψ method. The highest tensile residual strain was determined in a fusion line zone between the first as-deposited layer and a substrate. The microstructure of the first fusion line zone contained deformation twins and entangled dislocations generated by plastic flow under thermal expansion-contraction cycles.
Extracellular vesicles in cancer nanomedicine Tarasov, Vadim V.; Svistunov, Andrey A.; Chubarev, Vladimir N. ...
Seminars in cancer biology,
February 2021, 2021-02-00, 20210201, Letnik:
69
Journal Article
Recenzirano
To date, a lot of nanotechnological optitions are available for targeted drug delivery. Extracellular vesicles (EVs) are membrane structures that cells use for storage, transport, communication, and ...signaling. Recent research has focused on EVs as natural nanoparticles for drug delivery. This review sheds light on the application of EVs in cancer therapy, such as targeted chemotherapy, gene therapy, and vaccine development. Aspects of biogenesis, isolation, targeting, and loading of EVs are discussed in detail.
Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and ...flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.
Conformational switching is pervasively driven by protein interactions, particularly for intrinsically disordered binding partners. We developed a dually orthogonal fluorescence-based assay to ...monitor such events, exploiting environmentally sensitive fluorophores. This assay is applied to E3 ligase E6AP, as its AZUL domain induces a disorder-to-order switch in an intrinsically disordered region of the proteasome, the so-named Rpn10 AZUL-binding domain (RAZUL). By testing various fluorophores, we developed an assay appropriate for high-throughput screening of Rpn10:E6AP-disrupting ligands. We found distinct positions in RAZUL for fluorophore labeling with either acrylodan or Atto610, which had disparate spectral responses to E6AP binding. E6AP caused a hypsochromic shift with increased fluorescence of acrylodan-RAZUL while decreasing fluorescence intensity of Atto610-RAZUL. Combining RAZUL labeled with either acrylodan or Atto610 into a common sample achieved robust and orthogonal measurement of the E6AP-induced conformational switch. This approach is generally applicable to disorder-to-order (or
vice versa
) transitions mediated by molecular interactions.
An assay was developed to monitor a disorder-to-order switch in proteasome subunit Rpn10 that is induced by E6AP binding. Fluorophores acrylodan and Atto610 differentially measured E6AP-induced helicity in Rpn10 RAZUL, allowing for dual application.
PDF
