Profiling extra cellular matrix associated proteome of human fetal nucleus pulposus in search for regenerative targets.

Degeneration of the intervertebral disc is associated with a decrease in extra-cellular matrix (ECM) content due to an imbalance in anabolic and catabolic signaling. Our previous study profiled the core matrisome of fetal NP's and identified various proteins with anabolic potential for regenerative therapies. This study aims to complement those results by exploring ECM regulators, associated proteins and secreted factors of the fetal nucleus pulposus (NP). Proteomic data of 9 fetal, 7 healthy adults (age 22-79), and 11 degenerated NP's was analyzed. Based on the selection criteria, a total of 45 proteins were identified, of which 14 were uniquely expressed or upregulated in fetus compared to adult NP's. Pathway analysis with these proteins revealed a significant upregulation of one pathway and two biological processes, in which 12 proteins were involved. Prolyl 4 hydroxylase (P4HA) 1 and 2, Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) 1, and Heat shock protein 47 (SERPINH1) were involved in 'collagen biosynthesis' pathway. In addition, PLOD 1, SERPINH1, Annexin A1 and A4, CD109 and Galectin 3 (LGALS3) were all involved in biological process of 'tissue development'. Furthermore Annexin A1, A4 and A5, LGALS-3 and SERPINF1 were featured in 'negative regulation of cell death'. In conclusion, additionally to core ECM proteome, this study reveals ECM regulators and ECM affiliated proteins of interest to study for regenerative therapies, and their potential should be validated in future mechanistic experiments.

Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. SIGNIFICANCE: Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense in the patient tear. Negative regulators of these defense pathways were also found in patient tear indicating a fine balance between pathogen clearance and host tissue destruction during fungal infection depending upon the individual specific host - pathogen interaction. This understanding could be used to predict the progression and outcome of infection.

Data set of Aspergillus flavus induced alterations in tear proteome: Understanding the pathogen-induced host response to fungal infection.

Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled "Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection [1]" (Jeyalakhsmi Kandhavelu, Naveen Luke Demonte, Venkatesh Prajna Namperumalsamy, Lalitha Prajna, Chitra Thangavel, Jeya Maheshwari Jayapal, Dharmalingam Kuppamuthu, 2016). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.

Data set for the mass spectrometry based exoproteome analysis of Aspergillus flavus isolates.

Aspergillus flavus is one of the predominant causative organisms of mycotic keratitis in tropical parts of the world. Extracellular proteins are the earliest proteins that come in contact with the host and have a role in the infection process. Exoproteins of A. flavus isolated from infected cornea, sputum and a saprophyte were pooled and identified using high resolution mass spectrometry in order to get the total exoproteome from cultures isolated from different sources. A total of 637 proteins was identified from the pooled A. flavus exoproteome. Analysis based on GO annotations of the 637 identified proteins revealed that hydrolases form the predominant class of proteins in the exoproteome. Interestingly, a greater proportion of the exoproteins seem to be secreted through the non-classical pathways. This data represent the first in-depth analysis of the representative A. flavus exoproteome of a large set of isolates from distinct sources. This data have been deposited to the ProteomeXchange with identifier PXD001296.

Exoproteome of Aspergillus flavus corneal isolates and saprophytes: identification of proteoforms of an oversecreted alkaline protease.

Aspergillus flavus infects the human eye leading to keratitis. Extracellular proteins, the earliest proteins that come in contact with the host and virulence related exoproteins, were identified in the fungus isolated from infected cornea. Virulence of the corneal isolates was tested in the Galleria mellonella larvae model and those isolates showing higher virulence were taken for subsequent exoproteome analysis. High resolution two-dimensional electrophoresis and mass spectrometry were used to generate A. flavus exoproteome reference map as well as to profile most of the exoproteins. Analysis of the identified proteins clearly shows the major biological processes that they are involved in. Nearly 50% of the exoproteins possess catalytic activity and one of these, an alkaline serine protease (Alp1) is present in high abundance as well as multiple proteoforms. Many proteins in the A. flavus exoproteome have been shown to be virulence factors in other pathogens indicating the probable role for these proteins in the corneal infection as well. Interestingly, the majority of the exoproteins do not have secretory signal indicating that they are secreted through the non-classical pathway. Thus, this study provides a clue to the early strategies employed by the pathogen to establish an infection in an immunocompetent host. BIOLOGICAL SIGNIFICANCE: The outcome of a fungal infection in an immunocompetent human eye depends on the ability of the fungus to overcome the host defense and propagate itself. In this process, the earliest events with respect to the fungal proteins involved include the secretory proteins of the invading organism. As a first step towards understanding the role of the extracellular proteins, exoproteome profile of the fungal isolates was generated. The fungal isolates from cornea showed a distinct pattern of the exoproteome when compared to the saprophyte. Since corneal isolates also showed higher virulence in the insect larval model, presumably the proteins elaborated by the corneal isolates are virulence related. One of the abundant proteins is an alkaline serine protease and this protein exists as multiple proteoforms. This study reports the comprehensive profile of exoproteome and reveals proteins that are potential virulence factors.

ChiS histidine kinase negatively regulates the production of chitinase ChiC in Streptomyces peucetius.

Computational analysis of sequence homology of the chiSRC gene cluster, encoding a chitinase in Streptomyces peucetius, showed that the gene cluster could be a two-component regulon comprising a sensor kinase (chiS) and a response regulator (chiR). To prove that the ChiSRC is an authentic two-component system, the chiS gene was cloned and expressed in E.coli and the purified protein was used for biochemical analysis. In this report, we provide biochemical evidence to show that the sensor kinase encoded by chiS gene indeed is a histidine kinase capable of autophosphorylation and the histidine 144 residue of the ChiS protein is the phosphate acceptor. An insertion mutation at the chiS locus led to overproduction chitinase protein in S. peucetius implying that the chiC gene is negatively regulated by the two-component system.