bims-micpro Biomed News
on Discovery and characterization of microproteins
Issue of 2020‒06‒21
three papers selected by
Thomas Martinez
Salk Institute for Biological Studies

  1. J Biol Chem. 2020 Jun 17. pii: jbc.RA120.012948. [Epub ahead of print]
      The transport activity of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) in cardiac myocytes is modulated by an inhibitory interaction with a transmembrane peptide, phospholamban (PLB). Previous biochemical studies have revealed that PLB interacts with a specific inhibitory site on SERCA, and low-resolution structural evidence suggests that PLB interacts with distinct alternative sites on SERCA. High-resolution details of the structural determinants of SERCA regulation have been elusive because of the dynamic nature of the regulatory complex. In this study, we used computational approaches to develop a structural model of SERCA-PLB interactions in order to gain a mechanistic understanding of PLB-mediated SERCA transport regulation. We combined steered molecular dynamics (SMD) and membrane protein-protein docking experiments to achieve both a global search and all-atom force calculations to determine the relative affinities of PLB for candidate sites on SERCA. We modeled the binding of PLB to several SERCA conformations, representing different enzymatic states sampled during the calcium transport catalytic cycle. The results of the SMD and docking experiments indicated that the canonical PLB-binding site (comprising transmembrane helices M2, M4, and M9) is the preferred site. This preference was even more stringent for a super-inhibitory PLB variant. Interestingly, PLB-binding specificity became more ambivalent for other SERCA conformers. These results provide evidence for polymorphic PLB interactions with novel sites on M3 and with the outside of the SERCA helix M9. Our findings are compatible with previous physical measurements that suggest that PLB interacts with multiple binding sites, conferring dynamic responsiveness to changing physiological conditions.
    Keywords:  binding site; calcium ATPase; calcium transport; membrane biophysics; membrane protein; micropeptides; molecular docking; molecular dynamics; phospholamban; protein-protein interaction
  2. Cancer Immunol Res. 2020 Jun 19. pii: canimm.0886.2019. [Epub ahead of print]
      The success of cancer immunotherapy relies on the ability of cytotoxic T cells to specifically recognize and eliminate tumor cells based on peptides presented by HLA-I. Although the peptide epitopes that elicit the corresponding immune response often remain unidentified, it is generally assumed that neoantigens, due to tumor-specific mutations, are the most common targets. Here, we used a mass spectrometric approach to show an underappreciated class of epitopes that accounts for up to 15% of HLA-I peptides for certain HLA alleles in various tumors and patients. These peptides were translated from cryptic open reading frames in supposedly non-coding regions in the genome and were mostly unidentifiable with conventional computational analyses of mass spectrometry (MS) data. Our approach, Peptide-PRISM, identified thousands of such cryptic peptides in tumor immunopeptidomes. About 20% of these HLA-I peptides represented the C-terminus of the corresponding translation product, suggesting frequent proteasome-independent processing. Our data also revealed HLA-I allele-dependent presentation of cryptic peptides, with HLA-A*03 and HLA-A*11 presenting the highest percentage of cryptic peptides. Our analyses refute the reported frequent presentation of HLA peptides generated by proteasome-catalyzed peptide splicing (PCPS). Thus, Peptide-PRISM represents an important step towards comprehensive identification of HLA-I immunopeptidomes and reveals cryptic peptides as an abundant class of epitopes with potential relevance for novel immunotherapeutic approaches.
  3. mBio. 2020 Jun 16. pii: e01027-20. [Epub ahead of print]11(3):
      Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbor small upstream open reading frames (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in S. meliloti, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. Here, we provide evidence for a tryptophan-independent role of peTrpL in trans We found that peTrpL increases the resistance toward tetracycline, erythromycin, chloramphenicol, and the flavonoid genistein, which are substrates of the major multidrug efflux pump SmeAB. Coimmunoprecipitation with a FLAG-peTrpL suggested smeR mRNA, which encodes the transcription repressor of smeABR, as a peptide target. Indeed, upon antibiotic exposure, smeR mRNA was destabilized and smeA stabilized in a peTrpL-dependent manner, showing that peTrpL acts in the differential regulation of smeABR Furthermore, smeR mRNA was coimmunoprecipitated with peTrpL in antibiotic-dependent ribonucleoprotein (ARNP) complexes, which, in addition, contained an antibiotic-induced antisense RNA complementary to smeR In vitro ARNP reconstitution revealed that the above-mentioned antibiotics and genistein directly support complex formation. A specific region of the antisense RNA was identified as a seed region for ARNP assembly in vitro Altogether, our data show that peTrpL is involved in a mechanism for direct utilization of antimicrobial compounds in posttranscriptional regulation of multiresistance genes. Importantly, this role of peTrpL in resistance is conserved in other Alphaproteobacteria IMPORTANCE Leader peptides encoded by transcription attenuators are widespread small proteins that are considered nonfunctional in trans We found that the leader peptide peTrpL of the soil-dwelling plant symbiont Sinorhizobium meliloti is required for differential, posttranscriptional regulation of a multidrug resistance operon upon antibiotic exposure. Multiresistance achieved by efflux of different antimicrobial compounds ensures survival and competitiveness in nature and is important from both evolutionary and medical points of view. We show that the leader peptide forms antibiotic- and flavonoid-dependent ribonucleoprotein complexes (ARNPs) for destabilization of smeR mRNA encoding the transcription repressor of the major multidrug resistance operon. The seed region for ARNP assembly was localized in an antisense RNA, whose transcription is induced by antimicrobial compounds. The discovery of ARNP complexes as new players in multiresistance regulation opens new perspectives in understanding bacterial physiology and evolution and potentially provides new targets for antibacterial control.
    Keywords:  Agrobacterium tumefaciens ; Alphaproteobacteria ; Bradyrhizobium ; Sinorhizobium meliloti ; antibiotic resistance; antimicrobial compound; leader peptide; multidrug resistance; nucleoprotein complex; posttranscriptional RNA-binding protein; posttranscriptional control mechanisms; posttranscriptional regulation; ribonucleoprotein complex; transcription attenuator