Proteomic analysis of peripheral blood mononuclear cells isolated from patients with pulmonary tuberculosis: A pilot study from Zanzibar, Tanzania.

This study aimed at exploring the proteomic profile of PBMCs to predict treatment response in pulmonary tuberculosis (PTB). This was a pilot study conducted among 8 adult patients from Zanzibar, Tanzania with confirmed PTB. Blood samples were collected at baseline, at 2 months of treatment, and at the end of treatment at 6 months. Proteins were extracted from PBMCs and analyzed using LC-MS/MS based label free quantitative proteomics. Overall, 3,530 proteins were quantified across the samples, and 12 differentially expressed proteins were identified at both 2 months of treatment and at treatment completion, which were involved in cellular and metabolic processes, as well as binding and catalytic activity. Seven were downregulated proteins (HSPA1B/HSPA1A, HSPH1, HSP90AA1, lipopolysaccharide-binding protein, complement component 9, calcyclin-binding protein, and protein transport protein Sec31A), and 5 proteins were upregulated (SEC14 domain and spectrin repeat-containing protein 1, leucine-rich repeat-containing 8 VRAC subunit D, homogentisate 1,2-dioxygenase, NEDD8-activating enzyme E1 regulatory subunit, and N-acetylserotonin O-methyltransferase-like protein). The results showed that proteome analysis of PBMCs can be used as a novel technique to identify protein abundance change with anti-tuberculosis treatment. The novel proteins elucidated in this work may provide new insights for understanding PTB pathogenesis, treatment, and prognosis.

Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins.

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration.

Poly(L-lactide-co-ε-caprolactone) scaffolds were functionalised by 10 or 20 µg/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 µg/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.

Quantitative proteomic analyses of CD4+ and CD8+ T cells reveal differentially expressed proteins in multiple sclerosis patients and healthy controls.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune, neuroinflammatory disease, with an unclear etiology. However, T cells play a central role in the pathogenesis by crossing the blood-brain-barrier, leading to inflammation of the central nervous system and demyelination of the protective sheath surrounding the nerve fibers. MS has a complex inheritance pattern, and several studies indicate that gene interactions with environmental factors contribute to disease onset. METHODS: In the current study, we evaluated T cell dysregulation at the protein level using electrospray liquid chromatography-tandem mass spectrometry to get novel insights into immune-cell processes in MS. We have analyzed the proteomic profiles of CD4+ and CD8+ T cells purified from whole blood from 13 newly diagnosed, treatment-naive female patients with relapsing-remitting MS and 14 age- and sex-matched healthy controls. RESULTS: An overall higher protein abundance was observed in both CD4+ and CD8+ T cells from MS patients when compared to healthy controls. The differentially expressed proteins were enriched for T-cell specific activation pathways, especially CTLA4 and CD28 signaling in CD4+ T cells. When selectively analyzing proteins expressed from the genes most proximal to > 200 non-HLA MS susceptibility polymorphisms, we observed differential expression of eight proteins in T cells between MS patients and healthy controls, and there was a correlation between the genotype at three MS genetic risk loci and protein expressed from proximal genes. CONCLUSION: Our study provides evidence for proteomic differences in T cells from relapsing-remitting MS patients compared to healthy controls and also identifies dysregulation of proteins encoded from MS susceptibility genes.

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal (30LKKVMRR36) in the human enzyme. Substituting the residues KK31,32 and RR35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153.

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.

Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo.

A low dose of 1µg rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(l-lactide)-co-(ε-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects.

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine.

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukaemia cells.

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade) and the epoxomicin derivative PR-171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine- and cytokine-dependent proliferation of primary AML blasts when tested at nanomolar levels (0.1-100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin-like proteasome activity (showing a 20-fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR-171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells' constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose-dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.

Proteomic strategies for individualizing therapy of acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by accumulating myeloid precursor cells in the bone marrow, with approximately 2-3 months 50% survival if left untreated. With current treatment modalities the five years overall survival hardly exceeds 50%. Cytogenetics and molecular diagnostics guide the clinician to select individualized therapy in certain subsets of AML, achieving long-term survival above 70% of these cases. However, approximately half of the AML patients have no risk stratifying features, and early reports indicate that proteomic approaches may be utilized for disease classification as well as development of novel biomarkers related to prognosis, diagnosis, and choice of therapeutic regimen. Proteomics, here defined as the analysis of all proteins in a cell, in a cell compartment or in a signaling pathway, has probably its greatest potential in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. The major methodological challenges include detection sensitivity in a limited clinical material, a problem that in some cases can be solved through designated multiplexed protein assays based on single cells or cell extracts. In this review we will discuss pharmacoproteomic studies of drugs regulating leukemia specific targets like all-trans retinoic acid, histone deacetylase inhibitors, proteasome inhibitors and tyrosine kinase inhibitors, as well as studies on drug resistance and graft-versus-host studies during stem cell transplantations. These studies indicate new avenues in AML diagnostics, individualized therapy design and therapy response surveillance for the clinician.

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction.

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post-translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS-PAGE revealed high-molecular-mass forms in addition to the 36 kDa protomer. These forms were identified as poly-/multi-ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post-translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2-ubiquitin conjugates in the Triton X-100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin-binding protein.

The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N-terminal region of the enzyme.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL). The use of TRANSIL beads has allowed the determination of apparent dissociation constants (Kd) for the binding of the enzyme to negatively charged bilayers (Kd=230-380 microM, at pH 6.0-7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal alpha-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but alpha-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH.

Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.