Unit of Regulatory and Molecular Biology
Dr. Rameshwar Sharma, distinguished professor
Dr. Teresa Duda, professor
Dr. Rameshwar K. Sharma accepted the position of Distinguished Professor of Biochemistry and Molecular Biology in the Pennsylvania College of Optometry in mid-2006 and at the end of that year was joined by Dr. Teresa Duda (Professor of Biochemistry and Molecular Biology). Together they established the Unit of Regulatory and Molecular Biology with the aim of continuing studies on membrane guanylate cyclase signaling mechanism in the sensory neurons and cardiovascular system and to forma solid foundation of basic research at PCO-Salus University.
In a period spanning more than four decades, our research team has dedicated itself to the advancement of the field of membrane guanylate cyclase signal transduction in the vertebrate systems. This paved the way to novel and frontier venues. The cyclic GMP signaling was established as an intracellular hormonal signal transduction pathway and all the controversies on its non-existence were eliminated. The first membrane guanylate cyclase was purified to homogeneity and characterized in our laboratory. The protein was shown to be of dual activity; it was a receptor of the most hypotensive hormone ANF (atrial natriuretic factor) and the guanylate cyclase; therefore the name was coined ANF receptor guanylate cyclase, ANF-RGC (Paul et al., Science, 1987; Duda et al., PNAS 1992). This finding was revolutionary because the two, known at that time, second messenger signaling systems, cyclic AMP and inositol triphosphate, did not embody these characteristics. This discovery was the backbone of modern clinical medicine in treating hypertension. The drugs used are designed to keep the hormonal levels up and to generate cyclic GMP which, in turn relaxes smooth muscle and lowers blood pressure. ANF-RGC signal transduction was intriguing. Instead of GTP in G-Protein signaling, it was regulated by ATP through its ARM module (ARM) (Goraczniak et al., Biochem J. 1992; Duda et al., Mol Cell Biochem, 2002). After finding that the second natriuretic peptide receptor guanylate cyclase, CNP-RGC, is expressed in the retina (Duda et al., Biochemistry, 1993) we turned to the sensory neurons in order to decipher the role of membrane guanylate cyclase and cyclic GMP signaling in their function. In the course of these studies it became obvious that hormone-receptor guanylate cyclases constitute one subfamily, and the second subfamily comprises guanylate cyclases which are not receptors of any hormone but instead are regulated by intracellular calcium concentrations through specific small calcium sensing proteins. The discovery and molecular characterization of the rod outer segment guanylate cyclase, ROS-GC, was a land-mark event in the phototransduction field (Goraczniak et al., Biochem J. 1994). It filled in the gap on the identity of the source of cyclic GMP that serves as a second messenger of the LIGHT signal; and made it possible to explain the principles of phototransduction in molecular and physiological terms (Duda et al., Biochemistry 1996). It also impacted the core membrane guanylate cyclase field by branching the membrane guanylate cyclase family into two subfamilies: transducers of the hormone and of the intracellularly-generated calcium signals (Sharma, Mol. Cell Biochem, 2002). Our studies on calcium modulated guanylate cyclase signaling took additional direction with the demonstration that, besides phototransduction, it is biochemically linked with the transduction mechanisms of the inner segments of the retina, olfactory cilia and the olfactory bulb neurons (Venkataraman et al., Biochemistry, 2003; Duda et al., Biochemistry, 2001). Finally, we demonstrated that ROS-GC is a calcium-bimodal switch. This finding generated a new game-changing concept where theoretically ROS-GC could be modulated through two different modes of Ca2+ signaling: via calcium+-sensor GCAPs and S100B (Pozdnyakov et al., Biochemistry 1995; Duda et al., Biochemistry, 1996; Pozdnyakov et al., 1997).
In our present research conducted together with Senior Research Associate Dr. Alexandre Pertzev, we continue to be focused on membrane guanylate cyclase with special emphasis on its role in the cardiovascular and visual systems.
– From the discovery of ANF-RGC our laboratory has deciphered a major mechanism of blood pressure regulation: its product, cyclic GMP, is a critical vasorelaxant. We continue our studies on the natriuretic peptide hormones, ANF- and BNP receptor guanylate cyclase, ANF-RGC, and the premise that this signal transduction system is a major physiological regulator of the vasculatory tone. Its malfunction causes hypertension and leads to heart and kidney failure and also affects visual processes. Our primary goal is the decoding of the molecular principles of this transduction system: how ANF-RGC transduces the ANF signal and generates the production of its second messenger cyclic GMP and linking them with the vascular physiology and to determine how its aberrations lead to vascular pathologies. In the course of our studies through the concept-based analysis we identified a WTAPELL motif that controls the entire ANF-RGC signal transduction process (Duda et al., Mol Cell Biochem, 2009). We, then, predicted based on studies with recombinant systems, that this motif must be the one that controls the ANF-RGC-dependent blood pressure in the vasculature. To bring the findings to the physiological level we developed an in-ANF-RGC-gene WTAPELL deletion mouse model. This model was designed to link, for the first time, the specific signaling domain with the physiology of the ANF-RGC system. And indeed, the model confirmed our predictions, the mice expressing ANF-RGC with deleted the WTAPELL motif are hypertensive (Duda et al., Biochemistry 2012). This model has now been patented by Salus University.
In another ground-breaking discovery we demonstrated that ANF-RGC, in addition to the conventional mechanism of regulation by the natriuretic peptide hormones, is also susceptible to regulation by calcium ions. Calcium signals activation of ANF-RGC indirectly, through its sensor, neurocalcin delta. In calcium-bound-bound state neurocalcin delta interacts with and stimulates ANF-RGC catalytic domain to synthesize cyclic GMP. Importantly, because the calcium signaling via neurocalcin delta and ANF signaling, employ separate mechanisms, their effects on ANF-RGC activity are additive and together these two signals are capable of eliciting stronger response than each one alone. At the physiological level this phenomenon was confirmed through analyses of the neurocalcin delta- knock out mice. They have significantly increased blood pressure and are inflicted with cardiac myopathy (Duda et al., Biochemistry, 2012). Visual transduction. The discovery, by our group, of photoreceptor rod outer segment membrane guanylate cyclase (ROS-GC) allowed for the first time to explain the principles of phototransduction in molecular terms, the first step of visual transduction. It identified the guanylate cyclase that serves as a source of cyclic GMP, the second messenger of the LIGHT signal. Cloning of the ROS-GC gene made it possible to link its mutations to the various photoreceptor associated diseases: Leber’s congenital amaurosis (LCA1) and cone-rod dystrophy (CORD6) (Duda et al., Biochemistry 1999, 2000). Very recently, in collaboration with Dr. Makino’s group (initially at Harvard and presently at Boston University), we found an intriguing ROS-GC signaling pathway. This pathway is exclusively linked with the cones and is not involved with the rod physiology (Wen et al., Cell Physiol Biochem, 2012). It is anticipated that this finding will provide a basis of dissecting out the individual mechanisms of the rods and cones that are unique to themselves.
From its initial finding that ROS-GC membrane guanylate cyclase is a mono-modal Ca2+-transduction system linked exclusively with the phototransduction machinery to the successive finding that it embodies a remarkable bimodal Ca2+ signaling device, its widened transduction role in the general signaling mechanisms of the sensory neuron cells was envisioned. We proposed a theoretical concept where Ca2+-modulates ROS-GC and through it the levels of cyclic GMP which through a nearby cyclic nucleotide gated channel creates a hyper-or depolarized state in the neuron membrane. The generated electric potential then becomes a mode of transmission of the parent calcium signal. Calcium and ROS-GC are interlocked messengers in multiple sensory transduction mechanisms. This concept has been supported by the discovery of these types of linkages in the sensory transduction mechanisms of photoreceptor-“ON” bipolar cells, ganglion cells, olfactory receptor neurons, olfactory bulb neurons, pinealocytes and gustatory cells (Sharma and Duda, Frontiers in Molecular Neurosciences, 2014).
Our most recent finding is that ROS-GC1 is responsive to the bicarbonate signal; it is not the pH effect, rather bicarbonate binds to a specific motif of the ROS-GC and transduces the signal into generation of cyclic GMP (Duda et al., JBC, 2015). Preliminary findings suggest that this novel transduction system is present in a small number of mouse cones, and, importantly, is absent in the rods. This opens up a new ROS-GC-linked area of sensory transduction mechanism, where it is meant to communicate with the external atmospheric carbon dioxide. The carbon dioxide is sensed through the carbonic anhydrase enzyme, whose subtype has been detected in some cones, it converts CO2 to the bicarbonate, which, then, becomes the second messenger of CO2 and a signaling agent of ROS-GC1. Our research was and is funded by NSF and NIH.
We immensely enjoyed our fruitful long- lasting past collaborations with Drs. Ari Sitaramayya (PCO and Oakland University), Karl-Wilhelm Koch (Carl von Ossietzky University, Oldenburg, Germany) and Wolfgang Baehr (University of Utah). We are now involved in very productive collaborations with Drs. Clint L. Makino (Boston University) and Noga Vardi (University of Pennsylvania).
The results of the research conducted since joining PCO/Salus University were published in 29 peer-reviewed articles. During that time we also edited two membrane guanylate cyclase topic-oriented issues of Molecular and Cellular Biochemistry and Frontiers in Molecular Neurosciences and presently we organize a symposium on “Membrane Guanylate Cyclase, a Multimodal Cell Signaling Switch” for the 4th Global Experts Meeting on Neuropharmacology around the theme “Innovation and Complications in Neuropharmacological Studies” to be held September, 14-16, 2016 in San Antonio, Texas, USA.