PC1/3 KD Macrophages Exhibit Resistance to the Inhibitory Effect of IL-10 and a Higher TLR4 Activation Rate, Leading to an Anti-Tumoral Phenotype

During tumorigenesis, macrophages are recruited by tumors and orientated towards a pro-tumoral phenotype. One of the main anti-tumoral immunotherapy consists of their re-polarization in an anti-tumoral phenotype. We have demonstrated that the inhibition of proprotein convertase 1/3 combined with TLR4 activation in macrophages is a promising strategy. These macrophages display pro-inflammatory and anti-tumoral phenotypes. A hallmark is a stronger activation of the pro-inflammatory NFKB pathway. We believe that this can be explained by a modification of TLR4 expression at the cell surface or MYD88 cleavage since it exhibits a potential cleavage site for proprotein convertases. We tested these hypotheses through immunofluorescence and Western blot experiments. A proteomics study was also performed to test the sensitivity of these macrophages to IL-10. We demonstrated that these macrophages treated with LPS showed a quicker re-expression of TLR4 at the cell surface. The level of MYD88 was also higher when TLR4 was internalized. Moreover, these macrophages were resistant to the pro-tumoral effect of IL-10 and still produced pro-inflammatory factors. This established that the sensitivity to anti-inflammatory molecules and the length of TLR4 desensitization were reduced in these macrophages. Therefore, during antitumoral immunotherapy, a repeated stimulation of TLR4 may reactivate PC1/3 inhibited macrophages even in an anti-inflammatory environment.

Local and spatial factors determining HIV-1 protease substrate recognition

Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease–substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.

Characterization of a novel transcript of prostaglandin endoperoxide H synthase 1 with a tissue-specific profile of expression

The enzyme prostaglandin endoperoxide H synthase (PGHS) has a pivotal role in the prostanoid biosynthetic pathway because it catalyses the formation of prostaglandin H2 (PGH2), the common precursor of prostanoids. Two PGHS isoforms have been reported, PGHS-1 and PGHS-2, which have 61% identity (at the amino acid level) and 73% similarity (at the nucleotide level) between the two human enzymes. Transcription of the PGHS-1 gene leads to the formation of two transcripts (2.8 and 5.1 kb); two transcripts of 2.8 and 4.5 kb are produced from the PGHS-2 gene. By Northern blot analysis with the entire coding region of human PGHS-1, 2.8 and 5.1 kb transcripts as well as a novel 4.5 kb transcript were detected in the human megakaryoblastic cell line MEG-01. We designed a strategy to characterize the 4.5 kb PGHS transcript. Probes specific for each PGHS-1 and PGHS-2 were designed on the basis of the 3ʹ untranslated region (3ʹ UTR), where no similarity is present. The 4.5 kb transcript was detected only with the PGHS-1-specific 3ʹ UTR probes and not with the PGHS-2-specific 3ʹ UTR probe. To investigate the origin of the 4.5 kb PGHS-1 transcript, the remaining 947 bp of the 5.1 kb PGHS-1 transcript was generated by 3ʹ rapid amplification of cDNA ends (3ʹ RACE) and sequenced. A non-canonical polyadenylation signal (AAGAAA) located upstream of a potential cleavage site (CA) was found and could generate the 4.5 kb PGHS-1 transcript. Analysis of the sequence also produced several possible G/U-rich elements downstream of the potential cleavage site. An RNA dot-blot with 50 different human tissues was probed with the 4.5 and 5.1 kb PGHS-1-specific probes. A signal for the 4.5 kb PGHS-1 transcript was detected in the bladder and appendix. Signals of lower intensity were detected in the colon, bone marrow, small intestine, uterus, prostate, peripheral leucocyte, lymph node and stomach. In conclusion, our results suggest that the cell line MEG-01, the bladder and the appendix contain a new PGHS-1 transcript of 4.5 kb that can be produced from the PGHS-1 gene and we provide a better strategy for distinguishing PGHS-1 transcripts from PGHS-2.

Rapid and sensitive techniques for identification and analysis of ‘reactive-centre’ mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas

Novel procedures for structural analysis of the ‘reactive-centre‘ residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.