Kakoli Bose

Disorder and Structure in the Rab11 Binding Domain of Rab11- Family Interacting Protein 2 ,†,‡

Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for reuse at the cell surface. A recently identified family of Rab11 interacting proteins (FIP) includes FIP2. The C-terminal region of FIP2 is essential for colocalization with Rab11 on early endosomes and for enabling formation of higher-order oligomers. Rab11 binding and oligomerization of FIP2 are separable. Here we have determined the three-dimensional structure of the 40-residue coiled-coil oligomerization domain of FIP2 in the absence of Rab11 using NMR methods. The N-terminal half showed strong NOE cross-peaks and well-dispersed NMR resonances, whereas the C-terminal half had fewer NOE cross-peaks and less chemical shift dispersion. The 10 C-terminal residues were mostly disordered. The final structures of the dimer had favorable Ramachandran angles and a root-mean-square deviation of 0.59 +/- 0.13 A over superimposed backbone residues. The structure allows a comparison to a structure of FIP2 in complex with Rab11 that was determined crystallographically. In complex with Rab11, the C-terminal residues are not disordered but have a helical structure that predicts residual dipolar coupling constants that are incompatible with those measured on the unbound FIP2. In both structures, a histidine residue is found at the normally hydrophobic position of the heptad repeat of the coiled coil, and here we show its ionization destabilizes the coiled-coil structure. Together, these data allow us to build a model in which the binding of FIP family proteins to Rab11 can be described in terms of conformational changes and that suggests new modes of regulation.

Dual regulatory switch confers tighter control on HtrA2 proteolytic activity

High‐temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C‐terminal post‐synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens‐1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N‐terminus, as is evident from ‘inhibitor of apoptosis proteins’ binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N‐terminal ligand‐mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ‐independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site‐specific effector molecules.

Allosteric Regulation of Serine Protease HtrA2 through Novel Non-Canonical Substrate Binding Pocket

HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an in silico and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis.

Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2

HtrA2, a complex trimeric pyramidal mitochondrial serine protease that regulates critical biological functions and diseases, including apoptosis and cancer, is a promising therapeutic target. It promotes apoptosis through multiple pathways, complex mechanisms of which are still elusive. The existing model of activation that emphasizes relative intramolecular movements between C‐terminal PDZ and protease domains (PDZ‐protease collapse in inactive and resting states) has not been able to unambiguously demonstrate dynamics of its actions. Using structure‐guided design, molecular biology and protein biochemistry, we obtained various combinations of HtrA2 domains and mutants. Conformational changes and stability were characterized using molecular dynamics simulation and spectroscopic tools while functional enzymology delineated their roles in regulating enzyme catalysis. Quantitative Förster resonance energy transfer showed lesser intramolecular PDZ‐protease distance in trimeric HtrA2 compared to its inactive monomeric counterpart (~21 and ~22.3 Å, respectively, at 37°C). Our findings highlight importance of N‐terminal region, oligomerization, and intricate intermolecular PDZ‐protease interaction in proper active‐site formation, enzyme‐substrate complex stabilization, and hence HtrA2 functions. These observations redefine the existing activation model and showcase a unique example of how precise interdomain coordination, plasticity, and intermolecular contacts lead to distinct functional properties and hence provide new insights into HtrA2 structure, function, and dynamics.—Chaganti, L. K., Kuppili, R. R., Bose, K., Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2. FASEB J. 27, 3054–3066 (2013). www.fasebj.org

Molecular basis of death effector domain chain assembly and its role in caspase-8 activation

Assembly of a death‐inducing signaling complex is a key event in the extrinsic apoptotic pathway, enabling activation of the caspase cascade and subsequent cell death. However, the molecular events governing DISC assembly have remained largely elusive because of the lack of information on mechanism and specificity regulating the death effector domain (DED)‐DED interaction network. Using molecular modeling, mutagenesis, and biochemical and ex vivo experiments, we identified the precise binding interface and hot spots crucial for intermolecular DED chain assembly. Mutation of key interface residues (Leu42/Phe45) in procaspase‐8 DED‐A completely abrogated DED chain formation in HEK293 cells and prevented its association with FADD. A significant 2.6‐3.6‐fold reduction in procaspase‐8 activation was observed in functional cell‐death assays after substitution of the interfacial residues. Based on our results we propose a new model for DISC formation that refines the current understanding of the activation mechanism. Upon stimulation, FADD self‐associates weakly via reciprocal interaction between helices α1/α4 and α2/α3 of the DED to form an oligomeric signaling platform that provides a stage for the initial recruitment of procaspase‐8 through direct interaction with α1/α4 of DED‐A, followed by sequential interaction mediated by helices α2/α5 of DED‐B, to form the procaspase‐8 DED chain that is crucial for its activation and subsequent cell death.—Singh, N., Hassan, A., Bose, K. Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation. FASEB J. 30, 186‐200 (2016). www.fasebj.org

Apoptosis: Pathways, Molecules and Beyond

Programmed cell death or apoptosis manifests itself through a complex network of biochemical pathways and distinct morphological signatures. It is a natural phenomenon in multicellular organisms required to maintain tissue homeostasis through selective removal of ageing and unwanted cells. Impairment of this tightly regulated cellular process leads to various pathophysiological conditions including neurodegenerative disorders, ischemic damage, acquired immunodeficiency syndrome and cancer. Recognizing its immense therapeutic potential, a plethora of research endeavors has been undertaken in the past two decades that target molecules involved in apoptosis. Caspases, a conserved family of cysteinyl proteases that initiate and execute programmed cell death through extrinsic and intrinsic pathways are major focus of apoptosis research. However, study of molecules associated with lesser-known caspase-independent cell death is slowly gaining prominence, especially in cases where the traditional pathways fail to activate apoptosis. The goal of this chapter is to provide a broad overview of different apoptotic pathways, molecules involved and their crosstalk with special emphasis on proteases. This chapter also discusses different diseases associated with deregulation of apoptosis, current status on pre-clinical and clinical trials, their limitations and future prospects.

Cathepsins and HtrAs – Multitasking Proteases in Programmed Cell Death

Apoptosis is a crucial process in embryonic development, adult tissue homeostasis as well as in selective clearance of unwanted or infected cells. It is an energy dependent process generally initiated by cellular damage, stress or several endogenous and extracellular stimuli. During apoptosis, activation of specific proteases results in breakdown of cellular machinery which finally culminates into characteristic morphological and biochemical changes and hence death. For many years, it was believed that caspases were the only enzymes responsible for the proteolytic cascades in apoptosis. However, accumulating evidences indicate that cell death can occur in a programmed fashion in absence of caspase activation. For example, other proteases, such as cathepsins and HtrAs (high temperature requirement protease A), are also involved in the initiation and/or execution of the apoptosis. These proteases are capable of triggering mitochondrial dysfunction with subsequent caspase activation and cellular demise. Cathepsins, a group of proteases enclosed in the lysosomes, have a major role in apoptosis by triggering the death receptor as well as mitochondria-mediated apoptotic pathways. Apart from cathepsins, HtrAs also have a potential role in mediating apoptosis. HtrA family proteins are serine proteases that are involved in important physiological processes, including maintenance of mitochondrial homeostasis, apoptosis and cell signaling. They are involved in the development and progression of several diseases such as cancer, neurodegenerative disorders and arthritis. HtrA proteins are described as potential modulators of programmed cell death and chemotherapy-induced cytotoxicity. This chapter summarizes our current knowledge on the structural and functional aspects of these proteins, with an emphasis on their potential roles in apoptosis.

Design and characterization of an enhanced repressor of human papillomavirus E2 protein

Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure‐guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine‐glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2‐Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2‐responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.—Bose, K., Meinke, G., Bohm, A., Baleja, J. D. Design and characterization of an enhanced repressor of human papillomavirus E2 protein. FASEB J. 25, 2354–2361 (2011). www.fasebj.org

Calpains and Granzymes: Non-caspase Proteases in Cell Death

Proteolysis is the fundamental requirement of the process known as apoptosis or programmed cell death. Despite caspases being the primary molecules for apoptosis, other non-caspase proteases including calpains, granzymes, cathepsins, and the HtrA family of proteins also play pivotal roles in mediating and promoting cell death. This chapter is an attempt to discuss the different aspects of two of these non-caspase proteases: calpains and granzymes.

Proteases in Apoptosis: Protocols and Methods

Proteases in apoptosis have evolved as major drug targets in the past few decades. Development in this direction has been brought about by efficient design and refinement of the various platforms of protease assays. These can be broadly categorized into general assays, that characterize kinetics and biochemistry of apoptotic proteases, and the more specific assays devoted to discern proteases involved in apoptosis. Together, these two approaches comprise a flawless two-pronged approach to understand the role of proteases in apoptosis and their therapeutic applications. This chapter lays down a comprehensive account of different experimental procedures spanning the use of in vitro purified proteases to those that monitor enzyme activity and its apoptotic effect in fixed or live cells. In this regard, fluorescence based platforms are the workhorse of fast, accurate, easy-to-use and high throughput screening amenable procedures. Therefore, they form the majority of techniques, among others, covered in this chapter. Apart from the popular methods currently in use, this chapter also provides a bird’s eye view of the future of the protease assays with special mention of protease activatable prodrugs and protease engineering.