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Karyopherin beta 1 (Kpnβ1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP-MS) to identify Kpnβ1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnβ1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnβ1 and revealed enriched interactions between Kpnβ1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, CRM1, CCAR1 and FUBP1. Together, this study shows that Kpnβ1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the leading cause of cancer-related death worldwide. Surgical resection remains the definitive curative treatment for early-stage disease offering an overall 5-year survival rate of 62%. Despite careful case selection, a significant proportion of early-stage cancers relapse aggressively within the first year post-operatively. Identification of these patients is key to accurate prognostication and understanding the biology that drives early relapse might open up potential novel adjuvant therapies.Methods: We performed an unsupervised interrogation of >1600 serum-based autoantibody biomarkers using an iterative machine-learning algorithm.Results: We identified a 13 biomarker signature that was highly predictive for survivorship in post-operative early-stage lung cancer; this outperforms currently used autoantibody biomarkers in solid cancers. Our results demonstrate significantly poor survivorship in high expressers of this biomarker signature with an overall 5-year survival rate of 7.6%.Conclusions: We anticipate that the data will lead to the development of an off-the-shelf prognostic panel and further that the oncogenic relevance of the proteins recognised in the panel may be a starting point for a new adjuvant therapy. [ABSTRACT FROM AUTHOR]

Rheumatic heart disease (RHD) is a major cause of cardiovascular morbidity and mortality in low- and middle-income countries, where living conditions promote spread of group A β-haemolytic streptococcus. Autoimmune reactions due to molecular mimicry of bacterial epitopes by host proteins cause acute rheumatic fever (ARF) and subsequent disease progression to RHD. Despite knowledge of the factors that predispose to ARF and RHD, determinants of the progression to valvular damage and the molecular events involved remain incompletely characterised. This review focuses on altered protein expression in heart valves, myocardial tissue and plasma of patients with RHD and pathogenic consequences on RHD. Proteins mainly involved in structural organization of the valve matrix, blood homeostasis and immune response were altered due to RHD pathogenesis. Study of secreted forms of these proteins may aid the development of non-invasive biomarkers for early diagnosis and monitoring outcomes in RHD. Valve replacement surgery, the single evidence-based strategy to improve outcomes in severe RHD, is costly, largely unavailable in low- and middle-income countries (LMIC) and requires specialised facilities. When diagnosed early, penicillin prophylaxis may be used to delay progression to severe valvular damage. Echocardiography and cardiovascular magnetic resonance and the standard imaging tools recommended to confirm early diagnosis remain largely unavailable and inaccessible in most LMIC and both require expensive equipment and highly skilled persons for manipulation as well as interpretation of results. Changes in protein expression in heart valves and myocardium are associated with progressive valvular deformation in RHD. Understanding these protein changes should shed more light on the mechanisms of pathogenicity, while secreted forms of these proteins may provide leads towards a biomarker for non-invasive early detection of RHD. [ABSTRACT FROM AUTHOR]

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients. [ABSTRACT FROM AUTHOR]

Recent developments in the immuno-oncology field strongly support a role for the immune system in both the prevention and progression of melanoma. Melanoma is a highly immunogenic cancer, including its ability to induce tumour antigen-specific B cell and antibody responses through largely unknown mechanisms. This review considers likely hypothetical mechanisms by which anti-tumour surveillance detects pre-cancerous cells and by which immune (including B cell and antibody) responses may be elicited during malignancy. The review further considers potential pro- and anti-tumour functions of B cells and antibodies (including tertiary lymphoid structures) in both the tumour microenvironment and in circulation. Although the vast majority of studies have focused on T cells, recent evidence highlights the important roles of B cells in response to malignancy. B cells and antibodies are also discussed in the context of their potential utility as clinical biomarkers for various applications (as diagnostic, prognostic, therapeutic efficacy, and toxicity proxies), with a particular focus on protein microarray-based antibody detection and quantitation. Although the role of B cells in melanoma is incompletely understood, the measurement of circulating tumour-specific antibodies represents a promising avenue in the search for melanoma-relevant biomarkers. [ABSTRACT FROM AUTHOR]

Examines how a combination of design and necessity can rapidly evolve one enzyme in a biosynthetic pathway into another. Catalyst of the efficient synthesis of complex molecules; Evolution of enzymes in biological systems; Catalytic template for in vitro mutagenesis and recombination; Phosphoribosylanthranilate isomerase.

The effect of non-synonymous single nucleotide polymorphisms (SNPs) on cytochrome P450 (CYP450) drug metabolism is currently poorly understood due to the large number of polymorphisms, the diversity of potential substrates and the complexity of CYP450 function. Previously we carried out in silico studies to explore the effect of SNPs on CYP450 function, using in silico calculations to predict the effect of mutations on protein stability. Here we have determined the effect of eight CYP3A4 and seven CYP2C9 SNPs on the thermostability of proteins in solution to test these predictions. Thermostability assays revealed distinct CYP450 sub-populations with only 65-70% of wild-type CYP3A4 and CYP2C9 susceptible to rapid heat-induced P450 to P420 conversion. CYP3A4 mutations G56D, P218R, S222P, I223R, L373F and M445T and CYP2C9 mutations V76M, I359L and I359T were destabilising, increasing the proportion of protein sensitive to the rapid heat-induced P450 to P420 conversion and/or reducing the half-life of this conversion. CYP2C9 Q214L was the only stabilising mutation. These results corresponded well with the in silico protein stability calculations, confirming the value of these predictions and together suggest that the changes in thermostability result from destabilisation/stabilisation of the protein fold, changes in the haem-binding environment or effects on oligomer formation/conformation. [ABSTRACT FROM AUTHOR]

In the last 40 years only one new antitubercular drug has been approved, whilst resistance to current drugs, including rifampicin, is spreading. Here, we used the model organism Mycobacterium smegmatis to study mechanisms of phenotypic mycobacterial resistance, employing quantitative mass spectrometry-based proteomics to investigate the temporal effects of sub-lethal concentrations of rifampicin on the mycobacterial proteome at time-points corresponding to early response, onset of bacteriostasis and early recovery. Across 18 samples, a total of 3,218 proteins were identified from 31,846 distinct peptides averaging 16,250 identified peptides per sample. We found evidence that two component signal transduction systems (e.g. MprA/MprB) play a major role during initial mycobacterial adaptive responses to sub-lethal rifampicin and that, after dampening an initial SOS response, the bacteria supress the DevR (DosR) regulon and also upregulate their transcriptional and translational machineries. Furthermore, we found a co-ordinated dysregulation in haeme and mycobactin synthesis. Finally, gradual upregulation of the M. smegmatis-specific rifampin ADP-ribosyl transferase was observed which, together with upregulation of transcriptional and translational machinery, likely explains recovery of normal growth. Overall, our data indicates that in mycobacteria, sub-lethal rifampicin triggers a concerted phenotypic response that contrasts significantly with that observed at higher antimicrobial doses. [ABSTRACT FROM AUTHOR]