Proximity labeling techniques, such as APEX-MS, provide valuable insights into proximal interactome mapping; however, the verification of biotinylated peptides is not straightforward. With this as ...motivation, we present a new module integrated into PatternLab for proteomics to enable APEX-MS data interpretation by targeting diagnostic fragment ions associated with APEX modifications. We reanalyzed a previously published APEX-MS data set and report a significant number of biotinylated peptides and, consequently, a confident set of proximal proteins. As the module is part of the widely adopted PatternLab for proteomics software suite, it offers users a comprehensive, easy, and integrated solution for data analysis. Given the broad utility of the APEX-MS technique in various biological contexts, we anticipate that our module will be a valuable asset to researchers, facilitating and enhancing interactome studies. PatternLab’s APEX, including a usage protocol, is available at http://patternlabforproteomics.org/apex.
Pseudomonas fluorescens
CFBP2392 has been recognized as a potential biocontrol agent due to its ability to suppress damping-off and root rot disease. This isolate has antibacterial activity
in vitro
...as many other strains from the
Pseudomonas fluorescens
complex. In this work, the antibacterial and antifungal activity of the strain were explored. Dual culture assays evidenced the antifungal activity of the strain against different phytopathogens:
Alternaria
sp.,
Pythium ultimun
,
Fusarium oxysporum
, and
Rhizoctonia solani
. Purification of an antifungal fraction was performed by preparative HPLC from the chemical extraction of growth media. The fraction showed altered
R. solani
growth and ultrastructure. Transmission electron microscopy revealed the purified compound
induced
hypertrophied mitochondria, membranous vesicles, and a higher number of vacuoles in
R. salani
cytoplasm. In addition, co-cultivation of
P. fluorescens
CFBP2392 with
R. solani
resulted in an enlarged and deformed cell wall. To gain genomic insights on this inhibition, the complete genome of
P. fluorescens
CFBP2392 was obtained with Oxford Nanopore technology. Different biosynthetic gene clusters (BGCs) involved in specialized metabolites production including a lokisin-like and a koreenceine-like cluster were identified. In accordance with the putative BGCs identified, sequence phylogeny analysis of the MacB transporter in the lokisin-like cluster further supports the similarity with other transporters from the amphisin family. Our results give insights into the cellular effects of the purified microbial metabolite in
R. solani
ultrastructure and provide a genomic background to further explore the specialized metabolite potential.
(Mtb), the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine protein kinases, which play essential roles in cell growth, signal transduction, and pathogenesis. Protein kinase G ...(PknG) regulates the carbon and nitrogen metabolism by phosphorylation of the glycogen accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB) is involved in cell wall synthesis and cell shape, as well as phosphorylates GarA but at Thr22. While PknG seems to be constitutively activated and recognition of GarA requires phosphorylation in its unstructured tail, PknB activation is triggered by phosphorylation of its activation loop, which allows binding of the forkhead-associated domain of GarA. In the present work, we used molecular dynamics and quantum-mechanics/molecular mechanics simulations of the catalytically competent complex and kinase activity assays to understand PknG/PknB specificity and reactivity toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem essential for PknG binding and allow specificity for Thr21 phosphorylation. On the other hand, phosphorylated residues in PknB bind Arg26 in GarA and regulate its specificity for Thr22. We also provide a detailed analysis of the free energy profile for the phospho-transfer reaction and show why PknG has a constitutively active conformation not requiring priming phosphorylation in contrast to PknB. Our results provide new insights into these two key enzymes relevant for Mtb and the mechanisms of serine/threonine phosphorylation in bacteria.
Cellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic ...interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment.
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. ...Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.
•Plasma-derived exosomes from patients with CLL exhibit different protein cargo compositions depending on disease status and progression.•S100-A9 protein is overexpressed and S100-A9 cargo in exosomes activates NF-κB pathway in patients with CLL during disease progression.
Trypanothione synthetase (TryS) catalyses the synthesis of N
1
,N
8
-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in ...trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC
50
from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC
50
values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC
50
from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC
50
against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.
The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen ...Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.
Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related ...diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 μm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 μm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.
The Bovine Leukemia Virus (BLV) Envelope (Env) glycoprotein complex is instrumental in viral infectivity and shapes the host's immune response. This study presents the production and characterization ...of a soluble furin-mutated BLV Env ectodomain (sBLV-EnvFm) expressed in a stable S2 insect cell line. We purified a 63 kDa soluble protein, corresponding to the monomeric sBLV-EnvFm, which predominantly presented oligomannose and paucimannose N-glycans, with a high content of core fucose structures. Our results demonstrate that our recombinant protein can be recognized from specific antibodies in BLV infected cattle, suggesting its potential as a powerful diagnostic tool. Moreover, the robust humoral immune response it elicited in mice shows its potential contribution to the development of subunit-based vaccines against BLV.
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