Authors: Ping Li, Viktoria Bågenholm, Per Hägglund...

Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.P → E2 → E1 cycle. In the E2P and E2.P states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.P state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.

Topics: Cryoelectron Microscopy; Endoplasmic Reticulum; Adenosine Triphosphatases; Models, Molecular; Chaetomium; Protein Domains; Adenosine Triphosphate

PubMed: 39505844
DOI: 10.1038/s41467-024-53757-6

Review

Authors: Everly Conway de Macario, Masafumi Yohda, Alberto J L Macario...

Chaperonins are molecular chaperones that play critical physiological roles, but they can be pathogenic. Malfunctional chaperonins cause chaperonopathies of great interest within various medical specialties. Although the clinical-genetic aspects of many chaperonopathies are known, the molecular mechanisms causing chaperonin failure and tissue lesions are poorly understood. Progress is necessary to improve treatment, and experimental models that mimic the human situation provide a promising solution. We present two models: one prokaryotic (the archaeon ) with eukaryotic-like chaperonins and one eukaryotic (), both convenient for isolation-study of chaperonins, and report illustrative results pertaining to a pathogenic mutation of CCT5.

Topics: Archaeal Proteins; Bacterial Proteins; Chaperonins; Disease Susceptibility; Eukaryotic Cells; Fungal Proteins; Humans; Methanosarcinales; Molecular Chaperones; Mutation; Protein Conformation; Sulfolobales

PubMed: 30911678
DOI: 10.1038/s42003-019-0318-5

Authors: X W Wang, J Houbraken, J Z Groenewald...

During a study of indoor fungi, 145 isolates belonging to were cultured from air, swab and dust samples from 19 countries. Based on the phylogenetic analyses of DNA-directed RNA polymerase II second largest subunit (), β-tubulin (), ITS and 28S large subunit (LSU) nrDNA sequences, together with morphological comparisons with related genera and species, 30 indoor taxa are recognised, of which 22 represent known species, seven are described as new, and one remains to be identified to species level. In our collection, 69 % of the indoor isolates with six species cluster with members of the species complex, representing . The other indoor species fall into nine lineages that are separated from each other with several known chaetomiaceous genera occurring among them. No generic names are available for five of those lineages, and the following new genera are introduced here: with three indoor species, with one indoor species, with four indoor species, with seven indoor species and with two indoor species. The generic concept of is expanded to include and the chaetomium-like species (= ) in which two indoor species are included. The generic concept of is expanded to include several chaetomium-like taxa as well as one indoor species. is recognised as a distinct genus including two indoor taxa. According to this study, is the most abundant indoor species (74/145), followed by (17/145), (6/145) and (5/145). The morphological diversity of indoor as well as the morphological characteristics of the new genera are described and illustrated. This taxonomic study redefines the generic concept of and provides new insight into the phylogenetic relationships among different genera within .

PubMed: 28082757
DOI: 10.1016/j.simyco.2016.11.005

Authors: Charles Bou-Nader, Swati Gaikwad, Soheila Bahmanjah...

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNA. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNA. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2 was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2 of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Topics: Chaetomium; Protein Serine-Threonine Kinases; Histidine-tRNA Ligase; Stress, Physiological; Fungal Proteins; Crystallography, X-Ray; Models, Molecular; Protein Domains; Saccharomyces cerevisiae Proteins

PubMed: 39163341
DOI: 10.1073/pnas.2409628121

Authors: Christopher J Bley, Si Nie, George W Mobbs...

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].

Topics: Chaetomium; Cryoelectron Microscopy; Cytoplasm; Fungal Proteins; Humans; Molecular Chaperones; Nuclear Pore; Nuclear Pore Complex Proteins; Protein Conformation; RNA Transport; RNA, Messenger

PubMed: 35679405
DOI: 10.1126/science.abm9129

Review

Authors: Jinhua Chen, Wenzhou Zhang, Qingfeng Guo...

Chaetoglobosins belonging to cytochalasan alkaloids represent a large class of fungal secondary metabolites. To date, around 100 chaetoglobosins and their analogues have been isolated and identified over the years from a variety of fungi, mainly from the fungus . Studies have found that chaetoglobosins possess a broad range of biological activities, including antitumor, antifungal, phytotoxic, fibrinolytic, antibacterial, nematicidal, anti-inflammatory, and anti-HIV activities. This review will comprehensively summarize the biological activities and mechanisms of action of nature-derived chaetoglobosins.

PubMed: 32308719
DOI: 10.1155/2020/8574084

Authors: Yi Li, Paulina Fischer, Mengjiao Wang...

The spliceosome, a highly dynamic macromolecular assembly, catalyzes the precise removal of introns from pre-mRNAs. Recent studies have provided comprehensive structural insights into the step-wise assembly, catalytic splicing and final disassembly of the spliceosome. However, the molecular details of how the spliceosome recognizes and rejects suboptimal splicing substrates remained unclear. Here, we show cryo-electron microscopy structures of spliceosomal quality control complexes from a thermophilic eukaryote, Chaetomium thermophilum. The spliceosomes, henceforth termed B*, are stalled at a catalytically activated state but prior to the first splicing reaction due to an aberrant 5' splice site conformation. This state is recognized by G-patch protein GPATCH1, which is docked onto PRP8-EN and -RH domains and has recruited the cognate DHX35 helicase to its U2 snRNA substrate. In B*, DHX35 has dissociated the U2/branch site helix, while the disassembly helicase DHX15 is docked close to its U6 RNA 3'-end substrate. Our work thus provides mechanistic insights into the concerted action of two spliceosomal helicases in maintaining splicing fidelity by priming spliceosomes that are bound to aberrant splice substrates for disassembly.

Topics: Spliceosomes; Chaetomium; RNA Splicing; RNA Helicases; DEAD-box RNA Helicases; Fungal Proteins; RNA, Small Nuclear; Cryoelectron Microscopy

PubMed: 40016598
DOI: 10.1038/s41422-025-01084-w

Authors: Orsolya Kedves, Sándor Kocsubé, Teodóra Bata...

The genus is a frequently occurring fungal taxon world-wide. and -like species occur in indoor environments, where they can degrade cellulose-based building materials, thereby causing structural damage. Furthermore, several species of this genus may also cause adverse effects on human health. The aims of this research were to identify and -like strains isolated from indoor environments in Hungary and Finland, two geographically distant regions of Europe with drier and wetter continental climates, respectively, and to study their morphological and physiological properties, as well as their extracellular enzyme activities, thereby comparing the and -like species isolated from these two different regions of Europe and their properties. and -like strains were isolated from flats and offices in Hungary, as well as from schools, flats, and offices in Finland. Fragments of the translation elongation factor 1α (), the second largest subunit of RNA polymerase II () and β-tubulin () genes, as well as the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster were sequenced, and phylogenetic analysis of the sequences performed. Morphological examinations were performed by stereomicroscopy and scanning electron microscopy. Thirty-one sp. strains (15 from Hungary and 16 from Finland) were examined during the study. The most abundant species was in both countries. In Hungary, 13 strains were identified as , 1 as and 1 as . In Finland, 10 strains were 2 strains were , 2 were , and 2 isolates (SZMC 26527, SZMC 26529) proved to be representatives of a yet undescribed phylogenetic species from the closely related genus , which we formally describe here as the new species . Growth of the isolates was examined at different temperatures (4, 15, 20, 25, 30, 37, 35, 40, and 45 °C), while their extracellular enzyme production was determined spectrophotometrically.

PubMed: 34578165
DOI: 10.3390/pathogens10091133

Authors: Nathan W Polaske, Gary S Nichol, Bogdan Olenyuk...

The title compound, C(28)H(34)N(2)O(8)S(2), was synthesized as part of a project to develop synthetic routes to analogues of sporidesmins, a class of secondary metabolite produced by the filamentous fungi Chaetomium and Pithomyces sp. The complete molecule is generated by crystallographic inversion symmetry: the methoxy group is essentially coplanar with the benzene ring to which it is bonded, a mean plane fitted through the non-H atoms of the aromatic ring and the meth-oxy group having an r.m.s. deviation of 0.0140 Å. Similarly, the ester group is also essentially planar (r.m.s. deviation of a plane fitted through all non-H atoms is 0.0101 Å). There is only one independent C-H⋯O inter-action, which links together adjacent mol-ecules into a two-dimensional sheet in the bc plane.

PubMed: 21582859
DOI: 10.1107/S1600536809022211

Review

Authors: Zhihui Yuan, Yun Tian, Fulin He...

is a medicinal plant which contains abundant endophytes and various secondary metabolites. According to the literary about the information of endophytics from , , , , and were isolated from the root, stem, leaf, seed and bark of . The endophytics could produce lots of phytochemicals like flavonoids, terpenoids, and other compounds. These compounds have antibacteria, antioxidation, anticardiovascular, anticancer, antimicrobial and some novel functions. This paper set forth the development of active extracts isolated from endophytes of and will help to improve the resources of to be used in a broader field.

PubMed: 31728156
DOI: 10.1186/s13020-019-0271-8