Khushboo Gulati

Mechanistic and therapeutic overview of glycosaminoglycans: the unsung heroes of biomolecular signaling

Immune regulation is a complex biological signaling pathway in which several classes of biomolecules and small molecules play a complacent role to mediate this process. Glycoimmunology is a rapidly evolving research area that deals with the structure, binding interactions and immunological functions of glycans. Great deal of information regarding proteins and nucleic acids in molecular recognition events have been established owing to their well-established structural features and straight forward replication, transcription and translation principles. However considering the complexities of template free synthesis and structural heterogeneity, role of carbohydrates in immune regulation are still unsung to a large extent. In the current review, we illuminate the canonical structural features, emerging and significant pathophysiological functions of glycosaminoglycans (GAGs), the negatively charged linear carbohydrate molecules that are primarily present on all types of cell surfaces and extra cellular matrix. A snap shot of their association with protein counterparts of diversified protein families has been updated exclusively to provide mechanistic insights into their cellular signaling functions. Eventually, this review throws light on the recent biomedical/biotechnological advances of GAG based biomarkers, nutraceuticals, therapeutics, and nanocomposites for inflammatory, immune disorders and their invaluable contribution in tissue engineering.

Mechanistic insights into molecular evolution of species-specific differential glycosaminoglycan binding surfaces in growth-related oncogene chemokines

Chemokines are chemotactic cytokines involved in leucocyte trafficking to infected tissue. Growth-related oncogene (GRO) chemokines namely CXCL1, CXCL2 and CXCL3 are neutrophil activating chemokines sharing a conserved three-dimensional structure, but encompassing functional diversity due to gene duplication and evolutionary events. However, the evolutionary mechanisms including selection pressures involved in diversification of GRO genes have not yet been characterized. Here, we performed comprehensive evolutionary analysis of GRO genes among different mammalian species. Phylogenetic analysis illustrated a species-specific evolution pattern. Selection analysis evidenced that these genes have undergone concerted evolution. Seventeen positively selected sites were obtained, although the majority of the protein is under purifying selection. Interestingly, these positively selected sites are more concentrated on the C-terminal/glycosaminoglycan (GAG) binding and dimerization segment compared to receptor binding domain. Substitution rate analysis confirmed the C-terminal domain of GRO genes as the highest substituted segment. Further, structural analysis established that the nucleotide alterations in the GAG binding domain are the source of surface charge modulation, thus generating the differential GAG binding surfaces and multiple binding sites as per evolutionary pressure, although the helical surface is primordial for GAG binding. Indeed, such variable electrostatic surfaces are crucial to regulate chemokine gradient formation during a hosts defence against pathogens and also explain the significance of chemokine promiscuity.

Glycosaminoglycan-based resorbable polymer composites in tissue refurbishment

Regeneration of tissue structure with the aid of bioactive polymer matrices/composites and scaffolds for respective applications is one of the emerging areas of biomedical engineering. Recent advances in conjugated glycosaminoglycan (GAG) hybrids using natural and synthetic polymers have opened new avenues for producing a wide variety of resorbable polymer matrices. These hybrid scaffolds are low-immunogenic, highly biocompatible and biodegradable with incredible mechanical and tensile properties. GAG-based resorbable polymeric matrices are being exploited in migration of stem cells, cartilage and bone replacement/regeneration and production of scaffolds for various tissue engineering applications. In the current review, we will discuss the role of GAG-based resorbable polymer matrices in the field of regenerative medicine.

An Overview of Computational and Experimental Methods for Designing Novel Proteins.

BACKGROUND Unraveling the comprehensive networks of molecular signaling in various cellular processes and redesign/rewire them as per human wish is the ultimate dream of the biomedical researchers. Recent advances in the experimental and computational biophysics have provided us with enormous amount of protein sequences and a wide variety of structural information. Protein engineering is a fledging field and a creative process to design the target proteins or signaling networks with desirable structure and functions. OBJECTIVE Protein engineering has been a powerful tool in bioengineering for last couple of decades for generating vast numbers of useful enzymes/proteins that possess huge therapeutic and industrial potential. Now it is the high time to review the existing technologies and tune these methods for a desirable purpose as per the demand of biotechnological/biomedical applications. RESULTS Numerous engineering approaches have been developed to generate synthetic protein universe with desired specificity and enhanced performance in comparison to their natural counterparts. The current review provides a glimpse of several of the important computational and experimental methods that are being widely used under the categories of rational design, de novo design, directed evolution and combinatorial approach. CONCLUSIONS This review shed light on the technicalities, advantages and pitfalls of the existing methodologies along with their applications, recent patents obtained using the engineered proteins and the current and future perspectives of protein engineering techniques.

Deciphering the in vitro homo and hetero oligomerization characteristics of CXCL1/CXCL2 chemokines

Chemokines share the fundamental property of oligomerization and regulate leukocyte migration via interacting with glycosaminoglycans and G-protein coupled receptors. Our studies on murine neutrophil activating chemokines CXCL1(mKC)/CXCL2(MIP2) deciphered their differential homo oligomerization potentials and heterodimer forming capabilities, thus adding another layer of regulatory mechanism for leukocyte trafficking during infection/inflammation.

Molecular cloning and biophysical characterization of CXCL3 chemokine

CXCL3 is a neutrophil activating chemokine that belongs to GRO subfamily of CXC chemokines. GRO chemokine family comprises of three chemokines GRO α (CXCL1), GROβ (CXCL2), and GRO γ (CXCL3), which arose as a result of gene duplication events during the course of chemokine evolution. Although primary sequences of GRO chemokines are highly similar, they performs several protein specific functions in addition to their common property of neutrophil trafficking. However, the molecular basis for their differential functions has not well understood. Although structural details are available for CXCL1 and CXCL2, no such information regarding CXCL3 is available till date. In the present study, we have successfully cloned, expressed, and purified the recombinant CXCL3. Around 15mg/L of pure recombinant CXCL3 protein was obtained. Further, we investigated its functional divergence and biophysical characteristics such as oligomerization, thermal stability and heparin binding etc., and compared all these features with its closest paralog CXCL2. Our studies revealed that, although overall structural and oligomerization features of CXCL3 and CXCL2 are similar, prominent differences were observed in their surface characteristics, thus implicating for a functional divergence.

Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

Generating the Fancy Protein Basket with De Novo and Combinatorial Approaches

Under the umbrella of computational techniques, de novo approach occupies a unique role as this method is involved in designing the proteins from scratch. In the first part of the current chapter, we will discuss the principles and applications of the de novo approach along with negative designing technique. In the second part, we will elucidate the combinatorial approach of protein engineering, i.e., a hybrid approach to engineer proteins using both the experimental methods such as directed evolution techniques along with rational and de novo computational techniques. Furthermore, we will discuss various examples that glared the field of protein engineering under combinatorial approach.

Expanding the Synthetic Protein Universe by Guided Evolutionary Concepts

The genetic information content of a cell is maintained by the sequence composition of the DNA. The changes in the nucleotide content will potentially alter its transcriptional and translational events thus influencing the characteristics of the newly synthesized proteins. These nature’s alterations can be helpful in the evolution of proteins with novel/improved functionalities or they can contribute to the pathogenesis with loss of native functionalities. Unraveling the logistics of such a molecular evolutionary process is resourceful to strategically implement it for the benefit of the mankind through laboratory techniques. The laboratory process of synthesizing novel proteins in a constructive way through evolutionary guided principles is called “directed evolution”. This chapter will discuss various techniques, their strengths and pitfalls that are developed under the umbrella of directed evolution scheme.

Biotechnological and Biomedical Applications of Protein Engineering Methods

The fascinating field of protein engineering has provided breakthroughs by producing plethora of specifically engineered/rationally designed proteins with different functionalities and wide-scale applications in industrial, biotechnological, and pharmaceutical sectors. Many protein based therapeutics, vaccines, and scaffolds with greater safety, improved efficacy, reduced immunogenicity and improved delivery have been designed as novel biomedical formulations. In the present chapter, we will discuss the applications of engineered proteins in diversified fields of biotechnological and biomedical sciences including the areas of industrial, environmental, nanotechnology, biosensors, biomaterials, and biologics etc.