Journal: BMC Cell Biology

Article Title: Nuclear variants of bone morphogenetic proteins

doi: 10.1186/1471-2121-11-20

Figure Lengend Snippet: Inhibition of Bmp2 proprotein processing does not increase nuclear localization . (a) In vitro synthesis of radiolabeled Bmp2 preproprotein produced a 43 kDa protein (lane 1, large arrow). Incubation of this protein with recombinant furin for 1 or 3 hours generated a new protein band at 31 kDa, the predicted size of the free Bmp2 propeptide following proteolytic cleavage (lanes 2 and 3, small arrow). Preincubation of the furin with α 1 PDX, a serine protease inhibitor that blocks furin activity, prevented the formation of this band (lane 4). (b) Furin and α 1 PDX expression plasmids were each cotransfected with the wtBmp2/GFP fusion plasmid into RCS cells. Increasing furin expression did not significantly decrease nuclear localization of Bmp2/GFP, nor did inhibiting furin activity with α 1 PDX significantly increase nuclear localization. Mutation of the Bmp2 cleavage site to make it unrecognizable by furin or any related proprotein convertase (mtBmp2/GFP) also failed to significantly increase nuclear localization of Bmp2/GFP.

Article Snippet: Ten units of furin (New England BioLabs, Ipswich, MA) or furin plus 2 μM α 1 -PDX (Affinity BioReagents, Golden, CO) were preincubated for 30 min at room temperature.

Techniques: Inhibition, In Vitro, Produced, Incubation, Recombinant, Generated, Protease Inhibitor, Activity Assay, Expressing, Plasmid Preparation, Mutagenesis

Journal: Scientific Reports

Article Title: The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

doi: 10.1038/srep32371

Figure Lengend Snippet: ( A ) PsrP is organized into five domains: the N-terminal signal sequence (S) for extracellular translocation of PsrP, two putatively glycosylated serine rich repeat regions (SRR 1 and SRR 2 ), the binding region (BR) domain and the cell wall domain (CW). BR harbors two distinct sub-regions for KRT10 binding (black bar, residues 273–341) and self-oligomerization (black bar, residues 122–166), respectively. The sequence K 164 RRKR 168 is recognized by the human Furin protease. The three constructs BR 120–395 , BR 187–385 and BR 187–378 used within this study are displayed below the overall schematic representation of PsrP. In the mutated version of BR 120–395 (BR* 120–395 ), KRRKR was substituted to KSRKS. ( B ) A protease cleavage assay confirmed that furin recognized the KRRKR motif, but did not cleave in the presence of the furin protease inhibitor or when the KRRKR sequence was substituted to KSRKS. Cleavage was performed at room temperature (RT) and 37 °C for the indicated incubation times. The inhibitor was used at 100 μM and 250 μM, shown as plus signs in regular and bold format, respectively. ( C ) Distinct populations of BR 187–385 were eluted using Superdex 200 HiLoad 16/600 at elution column volumes (CV) of 89, 78, 71 and 66 mL corresponding to apparent molecular weights of 22, 55 and 90, 150 kDa, as well as higher oligomers. In this study, we show that the irreversibly associated dimer of BR 187–385 is formed through a domain swap mechanism (PDB: 5JUI, ) and that a low-affinity β-sheet dimer is created between two symmetry-related molecules in the previously determined crystal structure of the BR 187–385 monomer (PDB: 3ZGH, ). ( D ) BR* 120–395 was eluted from the same column at CV of 79 and 69 mL corresponding to apparent MW of 50 and 115 kDa. ( C , D ) When the isolated monomer and oligomer populations of BR 187–385 and BR* 120–395 were re-applied on analytical Superdex 200 HR10/30 columns, the monomer and oligomer populations were stable and did not interconvert between each other.

Article Snippet: Two units of Furin protease (New England Biolabs, P8077) were added to a total assay volume of 100 μL with a concentration of 0.7 mg/mL BR 120–395 and mutated BR* 120–395 in 20 mM HEPES, 500 mM NaCl, 10% glycerol, 1 mM CaCl 2 , pH 7.5.

Techniques: Sequencing, Translocation Assay, Binding Assay, Construct, Cleavage Assay, Protease Inhibitor, Incubation, Isolation