•Peptide markers from ribosomal proteins are diagnostic for lactic acid bacteria (LAB)•Reliable identification of lactic acid bacteria is performed by HR mass spectrometry.•LAB profiling by MS/MS is ...performed at genus, species and sub-species level.•The developed methodology allows the detection of LAB also in complex food matrices.
Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity.
In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/MS for LAB identification at genus, species and sub-species level by using the sequence regions 33–52 and 72–82 of the S16 ribosomal protein as proteotypic peptide markers.
The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices.
The highly conserved zinc finger CCCTC-binding factor (CTCF) regulates genomic imprinting and gene expression by acting as a transcriptional activator or repressor of promoters and insulator of ...enhancers. The multiple functions of CTCF are accomplished by co-association with other protein partners and are dependent on genomic context and tissue specificity. Despite the critical role of CTCF in the organization of genome structure, to date, only a subset of CTCF interaction partners have been identified. Here we present a large-scale identification of CTCF-binding partners using affinity purification and high-resolution LC-MS/MS analysis. In addition to functional enrichment of specific protein families such as the ribosomal proteins and the DEAD box helicases, we identified novel high-confidence CTCF interactors that provide a still unexplored biochemical context for CTCF's multiple functions. One of the newly validated CTCF interactors is BRG1, the major ATPase subunit of the chromatin remodeling complex SWI/SNF, establishing a relationship between two master regulators of genome organization. This work significantly expands the current knowledge of the human CTCF interactome and represents an important resource to direct future studies aimed at uncovering molecular mechanisms modulating CTCF pleiotropic functions throughout the genome.
Abstract
The selective oestrogen receptor (ER) degrader (SERD), fulvestrant, is limited in its use for the treatment of breast cancer (BC) by its poor oral bioavailability. Comparison of the orally ...bioavailable investigational SERD elacestrant, versus fulvestrant, demonstrates both drugs impact tumour growth of ER+ patient-derived xenograft models harbouring several
ESR1
mutations but that elacestrant is active after acquired resistance to fulvestrant. In cell line models of endocrine sensitive and resistant breast cancer both drugs impact the ER-cistrome, ER-interactome and transcription of oestrogen-regulated genes similarly, confirming the anti-oestrogenic activity of elacestrant. The addition of elacestrant to CDK4/6 inhibitors enhances the antiproliferative effect compared to monotherapy. Furthermore, elacestrant inhibits the growth of palbociclib-resistant cells. Lastly, resistance to elacestrant involves Type-I and Type-II receptor tyrosine kinases which are amenable to therapeutic targeting. Our data support the wider clinical testing of elacestrant.
Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. ...Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.
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•BAR enables to investigate the ERα proximal proteome in a spatial context.•Comparing epitope tag and endogenous antibodies for BAR.•Interrogating the ERα proximal proteome in endocrine-resistant BC models.•This study reports a high-confidence ERα proximal protein network.
Biotinylation by Antibody Recognition coupled with mass spectrometry is an effective approach to investigate the ERα proximal proteome. The described method successfully detected known ERα interactors and uncovered novel ERα-proximal partners involved in dynamic processes that are often challenging to detect with traditional protein interaction approaches.
A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform ...whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.
Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) ...produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP‐sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP‐glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild‐type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP‐sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state‐of‐the‐art quantitative proteomic approaches, as well as genetic and physiological information.
Systematic approach of metabolic engineering, combined with quantitative proteomics, as well as genetic and physiological information to identify bottlenecks for chondroitin‐like capsular polysaccaharide biosynthesis in E.coli K4 .
GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, ...MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells.
Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.
Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for ...imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20–37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTG DC GKY, GTG DC HVA, GSGT DC GSR, SQGS DC GSR) we obtained for all the proteins a cleavage efficiency of about 70–80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5–16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.
Secreted cytokines and growth factors play a key role in the modulation of stem cell proliferation, differentiation and survival. To investigate the interplay between the changes in their expression ...levels, we used the newly characterized human amniotic fluid derived‐mesenchymal progenitor MePR‐2B cell line differentiated to a neuro‐glial phenotype and exploited the very high sensitivity and versatility of magnetic beads‐based immunoassays. We found that a sub‐set of proteins, including the cytokines IL‐6, TNFα, IL‐15, IFNγ, IL‐8, IL‐1ra, MCP‐1/CCL2, RANTES and the growth factor PDGFbb, underwent a significant down‐regulation following neuro‐glial differentiation, whereas the expression levels of IL‐12 p70, IL‐5, IL‐7, bFGF, VEGF and G‐CSF were increased. The role of MCP‐1/CCL2, previously identified as a regulator of neural progenitor stem cell differentiation, has been further investigated at transcriptional level, revealing that both the chemokine and its receptor are co‐expressed in MePR‐2B cells and that are regulated upon differentiation, suggesting the presence of an autocrine and paracrine loop in differentiating cells. Moreover, we demonstrated that exogenous CCL2 is capable to affect neuro‐glial differentiation in MePR‐2B cells, thus providing novel evidences for the potential involvement of chemokine‐mediated signaling in progenitor/stem cells differentiation processes and fate specification.
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