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Mawla, G. D., Kamal, S. M., Cao, L. Y., Purhonen, P., Hebert, H., Sauer, R. T., . . . Römling, U. (2024). The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C. Journal of Biological Chemistry, 300(2), Article ID 105622.
Open this publication in new window or tab >>The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C
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2024 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 300, no 2, article id 105622Article in journal (Refereed) Published
Abstract [en]

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid–extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2’s MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

Place, publisher, year, edition, pages
Elsevier BV, 2024
Keywords
AAA+ ATPase, cytoplasmic linker, essential protease, M41 protease: periplasmic domain, Pseudomonas aeruginosa clone C, ssrA-tag
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-343477 (URN)10.1016/j.jbc.2023.105622 (DOI)38176647 (PubMedID)2-s2.0-85184070474 (Scopus ID)
Note

QC 20240216

Available from: 2024-02-15 Created: 2024-02-15 Last updated: 2024-02-16Bibliographically approved
Chen, G., Leppert, A., Poska, H., Nilsson, H., Alvira, C. P., Zhong, X., . . . Johansson, J. (2023). Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation. Communications Biology, 6(1), Article ID 497.
Open this publication in new window or tab >>Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation
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2023 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 6, no 1, article id 497Article in journal (Refereed) Published
Abstract [en]

BRICHOS domain oligomerization exposes three short hydrophobic motifs that are necessary for efficient chaperone activity against amorphous protein aggregation. ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

Place, publisher, year, edition, pages
Springer Nature, 2023
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-331231 (URN)10.1038/s42003-023-04883-2 (DOI)000992563300002 ()37156997 (PubMedID)2-s2.0-85158120481 (Scopus ID)
Note

QC 20230706

Available from: 2023-07-06 Created: 2023-07-06 Last updated: 2023-11-23Bibliographically approved
Kim, H., Lenoir, S., Helfricht, A., Jung, T., Karneva, Z. K., Lee, Y., . . . Song, J.-J. (2022). A pathogenic proteolysis-resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function. JCI Insight, 7(17), Article ID e154108.
Open this publication in new window or tab >>A pathogenic proteolysis-resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function
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2022 (English)In: JCI Insight, ISSN 2379-3708, Vol. 7, no 17, article id e154108Article in journal (Refereed) Published
Abstract [en]

Huntington's disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTT & UDelta;12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTT & UDelta;12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTT & UDelta;12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTT & UDelta;12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

Place, publisher, year, edition, pages
American Society for Clinical Investigation, 2022
National Category
Endocrinology and Diabetes Neurosciences Neurology
Identifiers
urn:nbn:se:kth:diva-320243 (URN)10.1172/jci.insight.154108 (DOI)000863210100001 ()35943803 (PubMedID)2-s2.0-85137662360 (Scopus ID)
Note

QC 20221019

Available from: 2022-10-19 Created: 2022-10-19 Last updated: 2022-10-19Bibliographically approved
Chen, G., Andrade-Talavera, Y., Zhong, X., Hassan, S., Biverstål, H., Poska, H., . . . Johansson, J. (2022). Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue. RSC Chemical Biology, 3(11), 1342-1358
Open this publication in new window or tab >>Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue
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2022 (English)In: RSC Chemical Biology, ISSN 2633-0679, Vol. 3, no 11, p. 1342-1358Article in journal (Refereed) Published
Abstract [en]

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pKa value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations. 

Place, publisher, year, edition, pages
Royal Society of Chemistry (RSC), 2022
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-328162 (URN)10.1039/d2cb00187j (DOI)000857166700001 ()36349220 (PubMedID)2-s2.0-85139932355 (Scopus ID)
Note

QC 20230602

Available from: 2023-06-02 Created: 2023-06-02 Last updated: 2023-11-23Bibliographically approved
Lee, E., Kang, C., Purhonen, P., Hebert, H., Bouazoune, K., Hohng, S. & Song, J.-J. (2021). A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity. Journal of Molecular Biology, 433(18), Article ID 167114.
Open this publication in new window or tab >>A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity
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2021 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 433, no 18, article id 167114Article in journal (Refereed) Published
Abstract [en]

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

Place, publisher, year, edition, pages
Elsevier BV, 2021
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-300764 (URN)10.1016/j.jmb.2021.167114 (DOI)000686349400007 ()34161779 (PubMedID)2-s2.0-85109198111 (Scopus ID)
Note

QC 20210917

Available from: 2021-09-02 Created: 2021-09-02 Last updated: 2022-06-25Bibliographically approved
Kumar, R. B., Purhonen, P., Hebert, H. & Jegerschöld, C. (2020). Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions. PLOS ONE, 15(7), Article ID e0228607.
Open this publication in new window or tab >>Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions
2020 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 15, no 7, article id e0228607Article in journal (Refereed) Published
Abstract [en]

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2020
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-279191 (URN)10.1371/journal.pone.0228607 (DOI)000552602700023 ()32645009 (PubMedID)2-s2.0-85087795152 (Scopus ID)
Note

QC 20200908

Available from: 2020-09-08 Created: 2020-09-08 Last updated: 2022-06-25Bibliographically approved
Chen, G., Andrade-Talavera, Y., Tambaro, S., Leppert, A., Nilsson, H. E., Zhong, X., . . . Johansson, J. (2020). Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro. Communications Biology, 3(1), Article ID 32.
Open this publication in new window or tab >>Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro
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2020 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 3, no 1, article id 32Article in journal (Refereed) Published
Abstract [en]

Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects.

Place, publisher, year, edition, pages
Nature Research, 2020
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-267767 (URN)10.1038/s42003-020-0757-z (DOI)000511413100001 ()31959875 (PubMedID)2-s2.0-85078256701 (Scopus ID)
Note

QC 20200303

Available from: 2020-03-03 Created: 2020-03-03 Last updated: 2024-03-15Bibliographically approved
Koulakiotis, N. S., Purhonen, P., Gikas, E., Hebert, H. & Tsarbopoulos, A. (2020). Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein. Scientific Reports, Article ID 74770.
Open this publication in new window or tab >>Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein
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2020 (English)In: Scientific Reports, ISSN 2045-2322, article id 74770Article in journal (Refereed) Published
Abstract [en]

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)—based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer’s Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Place, publisher, year, edition, pages
Nature Publishing Group, 2020
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-284859 (URN)10.1038/s41598-020-74770-x (DOI)000586485700008 ()33097779 (PubMedID)2-s2.0-85093921972 (Scopus ID)
Note

Correction in: Scientific Reports, Volume 11, Issue 1, December 2021, DOI: 10.1038/s41598-021-82907-9, Scopus id: 2-s2.0-85100277032

QC 20211116

Available from: 2020-11-04 Created: 2020-11-04 Last updated: 2022-07-11Bibliographically approved
Stsiapanava, A., Xu, C., Brunati, M., Zamora-Caballero, S., Schaeffer, C., Bokhove, M., . . . Jovine, L. (2020). Cryo-EM structure of native human uromodulin, a zona pellucida module polymer. EMBO Journal, 39(24), Article ID e106807.
Open this publication in new window or tab >>Cryo-EM structure of native human uromodulin, a zona pellucida module polymer
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2020 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 39, no 24, article id e106807Article in journal (Refereed) Published
Abstract [en]

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.

Place, publisher, year, edition, pages
EMBO, 2020
Keywords
cryo-electron microscopy, polymerization, uromodulin, zona pellucida, ZP domain
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-291166 (URN)10.15252/embj.2020106807 (DOI)000589518900001 ()33196145 (PubMedID)2-s2.0-85096769672 (Scopus ID)
Note

QC 20210304

Available from: 2021-03-04 Created: 2021-03-04 Last updated: 2022-09-19Bibliographically approved
Poska, H., Leppert, A., Tigro, H., Zhong, X., Kaldmae, M., Nilsson, H., . . . Johansson, J. (2020). Recombinant Bri3 BRICHOS domain is a molecular chaperone with effect against amyloid formation and non-fibrillar protein aggregation. Scientific Reports, 10(1), Article ID 9817.
Open this publication in new window or tab >>Recombinant Bri3 BRICHOS domain is a molecular chaperone with effect against amyloid formation and non-fibrillar protein aggregation
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2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 9817Article in journal (Refereed) Published
Abstract [en]

Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high beta-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer's disease associated amyloid-beta peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces A beta 42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.

Place, publisher, year, edition, pages
Springer Nature, 2020
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-278649 (URN)10.1038/s41598-020-66718-y (DOI)000543957900019 ()32555390 (PubMedID)2-s2.0-85086704392 (Scopus ID)
Note

QC 20200720

Available from: 2020-07-20 Created: 2020-07-20 Last updated: 2022-09-15Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-3220-9402

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