bims-micpro 2021-06-06 papers

Elife. 2021 Jun 02. pii: e65545. [Epub ahead of print]10

Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.

M'Lynn E Fisher, Elisa Bovo, Rodrigo Aguayo-Ortiz, Ellen E Cho, Marsha P Pribadi, Michael P Dalton, Nishadh Rathod, M Joanne Lemieux, L Michel Espinoza-Fonseca, Seth L Robia, Aleksey V Zima, Howard S Young.

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Keywords: biochemistry; chemical biology; none

DOI: https://doi.org/10.7554/eLife.65545

Int J Mol Sci. 2021 May 22. pii: 5476. [Epub ahead of print]22(11):

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing.

Bing Wang, Zhiwei Wang, Ni Pan, Jiangmei Huang, Cuihong Wan.

Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.

Keywords: de novo sequencing; non-ATG start codon; sORF-encoded peptides; sequence coverage; top-down

DOI: https://doi.org/10.3390/ijms22115476

PLoS Genet. 2021 Jun 01. 17(6): e1009585

Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.

Stephan Fuchs, Martin Kucklick, Erik Lehmann, Alexander Beckmann, Maya Wilkens, Baban Kolte, Ayten Mustafayeva, Tobias Ludwig, Maurice Diwo, Josef Wissing, Lothar Jänsch, Christian H Ahrens, Zoya Ignatova, Susanne Engelmann.

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.

DOI: https://doi.org/10.1371/journal.pgen.1009585