Methods and approaches to disease mechanisms using systems kinomics

All cellular functions, ranging from regular cell maintenance and homeostasis, specialized functions specific to cellular types, or generating responses due to external stimulus, are mediated by proteins within the cell. Regulation of these proteins allows the cell to alter its behavior under different circumstances. A major mechanism of protein regulation is utilizing protein kinases and phosphatases; enzymes that catalyze the transfer of phosphates between substrates [1]. Proteins involved in phosphate signaling are well studied and include kinases and phosphatases that catalyze opposing reactions regulating both structure and function of the cell. Kinomics is the study of kinases, phosphatases and their targets, and has been used to study the functional changes in numerous diseases and infectious diseases with aims to delineate the cellular functions affected. Identifying the phosphate signaling pathways changed by certain diseases or infections can lead to novel therapeutic targets. However, a daunting 518 putative protein kinase genes have been identified [2], indicating that this protein family is very large and complex. Identifying which enzymes are specific to a particular disease can be a laborious task. In this review, we will provide information on large-scale systems biology methodologies that allow global screening of the kinome to more efficiently identify which kinase pathways are pertinent for further study

Quantification of the Host Response Proteome after Herpes Simplex Virus Type 1 Infection

Viruses employ numerous host cell metabolic functions to propagate and manage to evade the host immune system. For herpes simplex virus type 1 (HSV1), a virus that has evolved to efficiently infect humans without seriously harming the host in most cases, the virus–host interaction is specifically interesting. This interaction can be best characterized by studying the proteomic changes that occur in the host during infection. Previous studies have been successful at identifying numerous host proteins that play important roles in HSV infection; however, there is still much that we do not know. This study identifies host metabolic functions and proteins that play roles in HSV infection, using global quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling of the host cell combined with LC–MS/MS. We showed differential proteins during early, mid and late infection, using both cytosolic and nuclear fractions. We identified hundreds of differentially regulated proteins involved in fundamental cellular functions, including gene expression, DNA replication, inflammatory response, cell movement, cell death, and RNA post-transcriptional modification. Novel differentially regulated proteins in HSV infections include some previously identified in other virus systems, as well as fusion protein, involved in malignant liposarcoma (FUS) and hypoxia up-regulated 1 protein precursor (HYOU1), which have not been identified previously in any virus infection

STEM in a Shoebox

<div> <div> <div>In the College of Engineering at Carnegie Mellon University, our faculty, staff and students are often asked to attend STEM events and visit schools to share STEM content with K-12 math and science classes. Requests are sometimes well in advance of the delivery date but can also be received at the last minute, with little time for adequate preparation. We are exploring a solution to this challenge that will serve to increase the participation of our STEM outreach volunteers and provide the recipients with a more complete STEM experience. The proposed solution is the advance preparation of stand-alone kits, complete with a scalable lesson plan, that will fit in a container with the approximate size of a ‘shoebox’ and will be stored and catalogued in the engineering and science library. <p>The original intent of the kit approach was to facilitate the College of Engineering’s collective inclusion of more stakeholders at the university (the library and maker space, for instance). In addition, teachers in the local school districts have had valuable input and look forward to the collaborative creation of additional kits. </p> <p>This paper is primarily about the process of creating a kit and scalable lesson plan that can be used for informal outreach as well as incorporated into larger broader impact initiatives. At Carnegie Mellon, this process has been developed by an interdisciplinary team from the College of Engineering, the Engineering and Science Library, the Engineering Research Accelerator, IdeATe (Integrative Design, Arts and Technology) Lab, and the Leonard Gelfand Center for Service Learning and Outreach. </p> </div> </div> </div

Quantification of the Host Response Proteome after Mammalian Reovirus T1L Infection

<div><p>All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular “transcriptome.” We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.</p> </div

Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells ▿ †

Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular “transcriptome.” Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways

Kinetics of reovirus growth and viral-induced cytopathology.

<p>Each of five different cell lines (L929, A549, HEK293, CaCo₂ and Hela) were infected at MOI  = 1 PFU/cell with T1L (a) or T3D (b). Cell lysates were harvested at 0, 24, 48 and 72hpi and titrated. Experiments were performed in triplicate; error bars represent standard error. Virus titers were greatest in the L929 and HEK293 cells for both virus strains. HEK293 (c) and L929 (d) cells were then re-analyzed as in (a) and (b) after infection at MOI  = 5 and at additional time points. Aliquots of the infections in (c) and (d) were also assessed for cell viability by trypan blue exclusion (e and f, respectively), with 100 μg/ml puromycin used as a positive cell killing control. Experiments were performed in duplicate; error bars represent standard error.</p

Top network functions generated using Ingenuity protein analysis for HEK293 cells infected with T1L reovirus at (a) 6hpi and (b) 24hpi.

<p>Graphs represent host cell functions with highest score (x-axis) based on the number of differentially regulated proteins observed in that network. The higher the score, the greater the number of proteins differentially regulated in that particular function network.</p

HEK293 proteins increased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p

HEK293 proteins decreased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p