MMP-2 is a novel histone H3 N-terminal protease necessary for myogenic gene activation.

BACKGROUND: Selective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown. RESULTS: In this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation. CONCLUSIONS: This study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.

Theta-Defensins Inhibit High-Risk Human Papillomavirus Infection Through Charge-Driven Capsid Clustering.

Persistent infection with high-risk human papillomavirus (hrHPV) genotypes results in a large number of anogenital and head and neck cancers worldwide. Although prophylactic vaccination coverage has improved, there remains a need to develop methods that inhibit viral transmission toward preventing the spread of HPV-driven disease. Defensins are a class of innate immune effector peptides that function to protect hosts from infection by pathogens such as viruses and bacteria. Previous work utilizing α and ß defensins from humans has demonstrated that the α-defensin HD5 is effective at inhibiting the most common high-risk genotype, HPV16. A third class of defensin that has yet to be explored are θ-defensins: small, 18-amino acid cyclic peptides found in old-world monkeys whose unique structure makes them both highly cationic and resistant to degradation. Here we show that the prototype θ-defensin, rhesus theta defensin 1, inhibits hrHPV infection through a mechanism involving capsid clustering that inhibits virions from binding to cell surface receptor complexes.

TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy.

Tumor necrosis factor superfamily member 14 (LIGHT) has been in pre-clinical development for over a decade and shows promise as a modality of enhancing treatment approaches in the field of cancer immunotherapy. To date, LIGHT has been used to combat cancer in multiple tumor models where it can be combined with other immunotherapy modalities to clear established solid tumors as well as treat metastatic events. When LIGHT molecules are delivered to or expressed within tumors they cause significant changes in the tumor microenvironment that are primarily driven through vascular normalization and generation of tertiary lymphoid structures. These changes can synergize with methods that induce or support anti-tumor immune responses, such as checkpoint inhibitors and/or tumor vaccines, to greatly improve immunotherapeutic strategies against cancer. While investigators have utilized multiple vectors to LIGHT-up tumor tissues, there are still improvements needed and components to be found within a human tumor microenvironment that may impede translational efforts. This review addresses the current state of this field.

Total chemical synthesis of murine ISG15 and an activity-based probe with physiological binding properties.

The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. Pilot experiments showed that the synthetic proteins were properly folded and recognized by endogenous enzymes. Our synthesis strategy allows the generation of other mISG15-based probes and reagents that can accelerate the research in this field.

A cross-species whole genome siRNA screen in suspension-cultured Chinese hamster ovary cells identifies novel engineering targets.

High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an in silico method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes, Rad21 and Chd4, whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.

Heterotetrameric annexin A2/S100A10 (A2t) is essential for oncogenic human papillomavirus trafficking and capsid disassembly, and protects virions from lysosomal degradation.

Human papillomavirus (HPV) entry into epithelial cells is independent of canonical endocytic pathways. Upon interaction with host cells, HPV establishes infection by traversing through an endocytic pathway that is clathrin- and caveolin-independent, but dependent on the annexin A2/S100A10 heterotetramer (A2t). We examined the contribution of monomeric annexin A2 (AnxA2) vs. A2t in HPV infection and endocytosis, and further characterized the role of these molecules in protein trafficking. We specifically show that cell surface A2t is not required for HPV attachment, and in the absence of A2t virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. These findings suggest that A2t is a central mediator of high-risk HPV intracellular trafficking post-entry and pre-viral uncoating.

Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants.

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbitpox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI-1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data-replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)-were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells.

Design of instrumentation and data-acquisition system for complex admittance measurement.

Instantaneous left ventricular volume measurements have been made for many years using a tetrapolar conductance catheter. The main objective is to determine the efficiency of the beating heart, using a tetrapolar catheter inserted in the left ventricle of transgenic mice. The effect of the parallel myocardium contribution must be removed from the total measurement. A dual-frequency technique involving 1 kHz and 100 kHz was chosen because it has been established that the imaginary part (the capacitive reactance) of the complex admittance of the cardiac muscle is much smaller in the lower frequency than at the higher frequency. The design involves generation of an accurate frequency source for both the frequencies careful selection of operational amplifiers for the current conversion stage so that the current is not too large to kill the mouse and that it is capable of performing at high frequencies. The band pass filter stage involved careful design with minimal overlap of the pass bands of both the channels. The overall circuit was designed so that there is minimal shift in the phase due to the circuit elements alone. Work also involved design of GPIB--based data acquisition system using LabVIEW and a digital oscilloscope for effective data acquisition even at high frequencies, which are normally limited by the sampling frequency. This data acquisition system is currently being used in laboratory studies in vivo.

Evidence of time-varying myocardial contribution by in vivo magnitude and phase measurement in mice.

Cardiac volume can be estimated by a conductance catheter system. Both blood and myocardium are conductive, but only the blood conductance is desired. Therefore, the parallel myocardium contribution should be removed from the total measured conductance. Several methods have been developed to estimate the contribution from myocardium, and they only determine a single steady state value for the parallel contribution. Besides, myocardium was treated as purely resistive or mainly capacitive when estimating the myocardial contribution. We question these assumptions and propose that the myocardium is both resistive and capacitive, and its contribution changes during a single cardiac cycle. In vivo magnitude and phase experiments were performed in mice to confirm this hypothesis.