The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits.

The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB-mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor-induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB-binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity.

Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism.

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.

The regulation of sequence specific NF-κB DNA binding and transcription by IKKß phosphorylation of NF-κB p50 at serine 80.

Phosphorylation of the NF-κB transcription factor is an important regulatory mechanism for the control of transcription. Here we identify serine 80 (S80) as a phosphorylation site on the p50 subunit of NF-κB, and IKKß as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNFα inducible expression of a subset of NF-κB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-κB binding (κB) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at κB sites with an adenine at the -1 position. Our analyses demonstrate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF-κB mediated transcription of pro-inflammatory genes. The regulation of a functional class of pro-inflammatory genes by the interaction of S80 phosphorylated p50 with a specific κB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses.

Communication, collaboration and contagion: "Virtualisation" of anatomy during COVID-19.

COVID-19 has generated a global need for technologies that enable communication, collaboration, education and scientific discourse whilst maintaining physical distance. University closures due to COVID-19 and physical distancing measures disrupt academic activities that previously occurred face-to-face. Restrictions placed on universities due to COVID-19 have precluded most conventional forms of education, assessment, research and scientific discourse. Anatomists now require valid, robust and easy-to-use communication tools to facilitate remote teaching, learning and research. Recent advances in communication, video conferencing and digital technologies may facilitate continuity of teaching and research activities. Examples include highly-interactive video conferencing technology, collaborative tools, social media and networking platforms. In this narrative review, we examine the utility of these technologies in supporting effective communication and professional activities of anatomists during COVID-19 and after.

Direct mechanical characterization of prostate tissue-a systematic review.

BACKGROUND: Direct mechanical characterization of tissue is the application of engineering techniques to biological tissue to ascertain stiffness or elasticity, which can change in response to disease states. A number of papers have been published on the application of these techniques to prostate tissue with a range of results reported. There is a marked variability in the results depending on testing techniques and disease state of the prostate tissue. We aimed to clarify the utility of direct mechanical characterization of prostate tissue in identifying disease states. METHODS: A systematic review of the published literature regarding direct mechanical characterization of prostate tissue was undertaking according to PRISMA guidelines. RESULTS: A variety of testing methods have been used, including compression, indentation, and tensile testing, as well as some indirect testing techniques, such as shear-wave elastography. There is strong evidence of significant stiffness differences between cancerous and non-cancerous prostate tissue, as well as correlations with prostate cancer stage. There is a correlation with increasing prostate stiffness and increasing lower urinary tract symptoms in patients with benign prostate hyperplasia. There is a wide variation in the testing methods and protocols used in the literature making direct comparison between papers difficult. Most studies utilise ex-vivo or cadaveric tissue, while none incorporate in vivo testing. CONCLUSION: Direct mechanical assessment of prostate tissue permits a better understanding of the pathological and physiological changes that are occurring within the tissue. Further work is needed to include prospective and in vivo data to aid medical device design and investigate non-surgical methods of managing prostate disease.

Regulation of muscle protein synthesis in an in vitro cell model using ex vivo human serum.

NEW FINDINGS: What is the central question of this study? Can medium conditioned by ex vivo human serum regulate muscle protein synthesis in skeletal muscle cells in vitro? What is the main finding and its importance? This study demonstrates that medium conditioned by ex vivo human serum can regulate muscle protein synthesis in skeletal muscle cells in vitro via the mammalian Target of Rapomycin (mTOR) pathway, and this can be regulated differentially by fed and fasted ex vivo human serum. ABSTRACT: Human serum embodies the integrated systemic response to any condition or perturbation, which may regulate muscle protein synthesis (MPS). Conditioning of medium with human serum represents a physiologically relevant method of regulating MPS in vitro. The primary purpose of the study was the development of a model using ex vivo human serum to condition medium and regulate MPS in in vitro skeletal muscle cells. Four young healthy men reported to the laboratory after an overnight fast and were fed with 0.33 g (kg body mass)-1 whey protein. Blood samples were taken before (Fasted) and 60 min postprandial (Fed). Fully differentiated C2C12 skeletal muscle cells were nutrient and serum deprived for 1 h and subsequently treated with medium conditioned with Fasted or Fed ex vivo human serum (20%) for 4 h. The MPS was measured using the surface sensing of translation technique and activation of mTOR, P70S6K and 4EBP1 by Western blot. Fasted and fed ex vivo human serum increased MPS (P < 0.05). Although a strong effect (ƞ2  = 0.36) for increased MPS in Fed relative to Fasted was observed, this was not statistically significant (P > 0.05). Activation of mTOR, P70S6K and 4EBP1 was significantly increased after treatment with Fed compared with Fasted ex vivo human serum (P < 0.05). Here, we developed and optimized the conditions for culture of C2C12 skeletal muscle cells, measurement of MPS and signalling in medium conditioned by ex vivo human serum. Furthermore, the functionality of the model was demonstrated by comparison of the response to medium conditioned by Fasted and Fed ex vivo human serum.

Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide.

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

Studying protein-protein interactions: progress, pitfalls and solutions.

Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success.

Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

NF-κB is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-κB occurs in the cytoplasm through the kinase activity of the IκB kinase complex, which leads to translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Protease-7 (USP7) as a regulator of NF-κB transcriptional activity. USP7 deubiquitination of NF-κB leads to increased transcription. Loss of USP7 activity results in increased ubiquitination of NF-κB, leading to reduced promoter occupancy and reduced expression of target genes in response to Toll-like- and TNF-receptor activation. These findings reveal a unique mechanism controlling NF-κB activity and demonstrate that the deubiquitination of NF-κB by USP7 is critical for target gene transcription.

Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

Inhibition of transcription by B cell Leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.

B cell leukemia 3 (Bcl-3) is an essential negative regulator of NF-κB during Toll-like receptor and TNF receptor signaling. Bcl-3 also interacts with a number of transcriptional regulators, including homodimers of the NF-κB p50 subunit. Deletion of Bcl-3 results in increased NF-κB p50 ubiquitination and proteasomal degradation and increased inflammatory gene expression. We employed immobilized peptide array technology to define a region of p50 required for the formation of a Bcl-3·p50 homodimer immunosuppressor complex. Our data demonstrate that amino acids 359-361 and 363 of p50 are critical for interaction with Bcl-3 and essential for Bcl-3-mediated inhibition of inflammatory gene expression. Bcl-3 is unable to interact with p50 when these amino acids are mutated, rendering it incapable of inhibiting the transcriptional activity of NF-κB. Bcl-3 interaction-defective p50 is hyperubiquitinated and has a significantly reduced half-life relative to wild-type p50. Nfkb1(-/-) cells reconstituted with mutated p50 precursor p105 are hyperresponsive to TNFα stimulation relative to wild-type p105, as measured by inflammatory gene expression. Mutant p105 recapitulates a Bcl3(-/-) phenotype. This study demonstrates that interaction with p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3 and further highlights the importance of p50 homodimer stability in the control of NF-κB target gene expression.

Direct interaction between scaffolding proteins RACK1 and 14-3-3ζ regulates brain-derived neurotrophic factor (BDNF) transcription.

RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions.

Serine phosphorylation of the insulin-like growth factor I (IGF-1) receptor C-terminal tail restrains kinase activity and cell growth.

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3ß in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3ß knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3ß restrains kinase activity and regulates receptor trafficking and signaling.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris.

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain 'adaptor' protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

RACK1 to the future--a historical perspective.

This perspective summarises the first and long overdue RACK1 meeting held at the University of Limerick, Ireland, May 2013, in which RACK1's role in the immune system, the heart and the brain were discussed and its contribution to disease states such as cancer, cardiac hypertrophy and addiction were described. RACK1 is a scaffolding protein and a member of the WD repeat family of proteins. These proteins have a unique architectural assembly that facilitates protein anchoring and the stabilisation of protein activity. A large body of evidence is accumulating which is helping to define the versatile role of RACK1 in assembling and dismantling complex signaling pathways from the cell membrane to the nucleus in health and disease. In this commentary, we first provide a historical perspective on RACK1. We also address many of the pertinent and topical questions about this protein such as its role in transcription, epigenetics and translation, its cytoskeletal contribution and the merits of targeting RACK1 in disease.

Collagen-I influences the post-translational regulation, binding partners and role of Annexin A2 in breast cancer progression.

Introduction: The extracellular matrix (ECM) has been heavily implicated in the development and progression of cancer. We have previously shown that Annexin A2 is integral in the migration and invasion of breast cancer cells and in the clinical progression of ER-negative breast cancer, processes which are highly influenced by the surrounding tumor microenvironment and ECM. Methods: We investigated how modulations of the ECM may affect the role of Annexin A2 in MDA-MB-231 breast cancer cells using western blotting, immunofluorescent confocal microscopy and immuno-precipitation mass spectrometry techniques. Results: We have shown that the presence of collagen-I, the main constituent of the ECM, increases the post-translational phosphorylation of Annexin A2 and subsequently causes the translocation of Annexin A2 to the extracellular surface. In the presence of collagen-I, we identified fibronectin as a novel interactor of Annexin A2, using mass spectrometry analysis. We then demonstrated that reducing Annexin A2 expression decreases the degradation of fibronectin by cancer cells and this effect on fibronectin turnover is increased according to collagen-I abundance. Discussion: Our results suggest that Annexin A2's role in promoting cancer progression is mediated by collagen-I and Annexin A2 maybe a therapeutic target in the bi-directional cross-talk between cancer cells and ECM remodeling that supports metastatic cancer progression.

RACK1, A multifaceted scaffolding protein: Structure and function.

The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the ß subunit of G-proteins (Gß). RACK1 adopts a seven-bladed ß-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.

Collagen and fibronectin promote an aggressive cancer phenotype in breast cancer cells but drive autonomous gene expression patterns.

Understanding how various pathologies of breast cancer respond to their environment may be imperative in the creation of novel therapeutic targets. Central to the organisation and behaviour of cells within the tumour microenvironment is the extracellular matrix (ECM), a meshwork of fibrous proteins and glycoproteins that directly influences cell behaviour and the bioavailability of signalling molecules. Our appreciation on how the composition of the ECM can influence cancer behaviour has evolved significantly and although we are highly cognisant of the dramatic impact the ECM can have on cancer cell behaviour, we continue to neglect this during diagnosis and treatment. In the following study, we aimed to identify how three breast cancer cell lines respond functionally and genetically to common components of the ECM. Using real time and end point assays we have identified similar patterns of behaviour among the three breast cancer cell lines in response to commonly found ECM components of the breast. Using a selected gene panel, we have been able to identify cell line specific changes in gene differentiation when breast cancer cells are in contact with these elements. Although the response of our cells to these elements differ at the genetic level, their functional responses are consistent. This work adds to the growing arguments that highlight a need for histologically assessing ECM composition of breast tumours. In particular monitoring of fibrous protein deposition at the site of malignancy could provide critical information during clinical assessment influencing disease prognosis and treatment decisions for breast cancer patients.

Proposed hypothesis and rationale for association between mastitis and breast cancer.

Breast cancer is amongst the most common forms of cancer, is predominantly a woman's illness, and is the most frequently reported invasive cancer in women worldwide (Bray et al., 2018). Varying risk factors have been identified, including genetics, family history, lifestyle, age and the use of hormone replacement therapy. Mastitis, also predominantly a woman's illness, is an inflammatory condition of the breast that, despite being an inflammation-related condition, is not currently considered a risk factor for breast cancer. This appears counterintuitive as epidemiological studies have identified chronic inflammation as a contributor to cancer risk, for example in gastric, oesophageal and colon cancers (Lin et al., 2016; Qadri et al., 2014; Principe et al., 2017). Previous reports have focused on women hospitalised for mastitis, and most commonly on puerperal mastitis, perhaps underestimating the relationship between breast cancer and non-lactational mastitis. Our hypothesis, based on systematic review, suggests that a longitudinal study of this disease, affecting women predominantly, is warranted.

Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers.

When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.