Sensitive Identification of Known and Unknown Protease Activities by Unsupervised Linear Motif Deconvolution

AbstractThe hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

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Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200706034 ◽

2007 ◽

Vol 179 (5) ◽

pp. 935-950 ◽

Cited By ~ 100

Author(s):

K.G. Suresh Kumar ◽

Hervé Barriere ◽

Christopher J. Carbone ◽

Jianghuai Liu ◽

Gayathri Swaminathan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Interferon Α ◽

Type I ◽

Lysosomal Degradation ◽

Linear Motif ◽

Site Specific ◽

Receptor Endocytosis ◽

Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

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Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors

Open Biology ◽

10.1098/rsob.150063 ◽

2015 ◽

Vol 5 (8) ◽

pp. 150063 ◽

Cited By ~ 6

Author(s):

Stephen Short ◽

Tessa Peterkin ◽

Matthew Guille ◽

Roger Patient ◽

Colin Sharpe

Keyword(s):

Alternative Splicing ◽

Gene Sequence ◽

Species Differences ◽

Single Point ◽

Stem Cell Differentiation ◽

Single Point Mutation ◽

Linear Motif ◽

Cellular Context ◽

Alternatively Spliced ◽

Linear Motifs

Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus , alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment.

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BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

10.1101/2020.06.12.148254 ◽

2020 ◽

Author(s):

Laura Bel Borja ◽

Flavie Soubigou ◽

Samuel J.P. Taylor ◽

Conchita Fraguas Bringas ◽

Jacqueline Budrewicz ◽

...

Keyword(s):

Kinase Domain ◽

Meiotic Spindle ◽

Dependent Manner ◽

Female Meiosis ◽

Linear Motif ◽

Interaction Domain ◽

C Elegans ◽

Chromosome Congression ◽

B Subunits ◽

Meiosis I

ABSTRACTProtein Phosphatase 2A (PP2A) is an heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits with various key roles during cell division. While A and C subunits form the core enzyme, the diversity generated by interchangeable B subunits dictates substrate specificity. Within the B subunits, B56-type subunits play important roles during meiosis in yeast and mice by protecting centromeric cohesion and stabilising the kinetochore-microtubule attachments. These functions are achieved through targeting of B56 subunits to centromere and kinetochore by Shugoshin and BUBR1. In the nematode Caenorhabditis elegans (C. elegans) the closest BUBR1 ortholog lacks the B56 interaction domain and the Shugoshin orthologue is not required for normal segregation during oocyte meiosis. Therefore, the role of PP2A in C. elegans female meiosis is not known. Here, we report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. Specifically, B56 subunits PPTR-1 and PPTR-2 associate with chromosomes during prometaphase I and regulate chromosome congression. The chromosome localization of B56 subunits does not require shugoshin orthologue SGO-1. Instead we have identified the kinase BUB-1 as the key B56 targeting factor to the chromosomes during meiosis. PP2A BUB-1 recruits PP2A:B56 to the chromosomes via dual mechanism: 1) PPTR-1/2 interacts with the newly identified LxxIxE short linear motif (SLiM) within a disordered region in BUB-1 in a phosphorylation-dependent manner; and 2) PPTR-2 can also be recruited to chromosomes in a BUB-1 kinase domain-dependent manner. Our results highlight a novel, BUB-1-dependent mechanism for B56 recruitment, essential for recruiting a pool of PP2A required for proper chromosome congression during meiosis I.

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A Comparative Study of Short Linear Motif Compositions of the Influenza A Virus Ribonucleoproteins

PLoS ONE ◽

10.1371/journal.pone.0038637 ◽

2012 ◽

Vol 7 (6) ◽

pp. e38637 ◽

Cited By ~ 3

Author(s):

Chu-Wen Yang

Keyword(s):

Comparative Study ◽

Influenza A Virus ◽

Influenza A ◽

Linear Motif

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Evolutionary conservation of the intrinsic disorder-based Radical-Induced Cell Death1 hub interactome

Scientific Reports ◽

10.1038/s41598-019-55385-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Lise Friis Christensen ◽

Lasse Staby ◽

Katrine Bugge ◽

Charlotte O’Shea ◽

Birthe B. Kragelund ◽

...

Keyword(s):

Transcription Factor ◽

Stress Responses ◽

Plant Stress ◽

Three Dimensional ◽

Intrinsic Disorder ◽

Dimensional Structure ◽

Linear Motif ◽

Intrinsically Disordered ◽

Helix Formation ◽

Α Helix

AbstractRadical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.

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