Featured Researchers/Lab

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.

Past Research

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).

Present Research

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.

Cardiovascular system – 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.

Dr. Alex Dizhoor moved his laboratory from Wayne State University to the Pennsylvania College of Optometry in 2002 to conduct studies in molecular biology, pharmacology and congenital diseases of photoreceptors. He was awarded the (then) newly established Hafter Family Chair in Pharmacology.

The main field of study in my laboratory is signal transduction in retinal rods and cones and its link to congenital blinding disorders. As the first step in seeing, rod and cone photoreceptors in the retina respond to light by generating an electrical pulse and convert it into a chemical synaptic input to other neurons, which, after processing through the secondary and tertiary neurons, ends up in the visual cortex of the brain. What carries the signal transduction in photoreceptors is a messenger molecule called cyclic GMP (cGMP) produced by enzyme called retinal guanylyl cyclase, RetGC. The RetGC becomes activated by calcium binding proteins called GCAPs (guanylyl cyclase activating proteins). In the dark, GCAPs bind calcium and suppress RetGC activity. Light triggers a rapid decay of cGMP, which causes calcium concentrations in photoreceptors to fall. This activates GCAP/RetGCs complex and helps photoreceptors to quickly recover from excitation and adapt to different levels of illumination. In addition to its importance for the biology of vision, proper regulation of RetGC by GCAPs is also essential for the survival of photoreceptors. Mutations in RetGC and GCAPs can cause either deficiency or excessive production of cGMP and consequently incapacitate photoreceptors or trigger their self-destruction, both leading to congenital blindness. The directions of the studies ongoing in my lab reflect the complexity of the biochemical, physiological and pathogenic processes surrounding the mechanisms of cGMP regulation in photoreceptors by GCAPs and RetGC.

In the past, when I worked at University of Washington and Wayne State University, I, together with Elena and my other colleagues, made a number of major contributions to the field. We were the first who identified the GCAP-regulated guanylyl cyclase in photoreceptors, discovered one of the GCAP proteins and another retinal calcium sensor protein called recoverin (Dizhoor et al., 1993; 1994, 1995; Lowe et al., 1995). We were also the first who demonstrated a direct biochemical link between mutations in GCAP and the onset of congenital retinal degeneration (Dizhoor et al., 1998). After I moved my laboratory to PCO, together with Elena, Igor, and our colleagues at Harvard, UCLA and NIH we were also the first who replicated in transgenic mice mutations in GCAP that caused congenital photoreceptor degeneration in human patients (Olshevskaya et al., 2004; Woodruff et al., 2007). We demonstrated in those genetic models biochemical and physiological changes in the retina that lead to retinal degeneration and thus confirmed the predictions from our in vitro studies of the mutated proteins.

My work as a PI and the researcher also contributed in establishing a new direction in modern molecular physiology and photobiology now known as optogenetics - artificial modulation of electrical activity of the cells by light- dependent ion channels. Together with the laboratory of Zho-Hua Pan in Wayne State University, we were the first who restored light sensitivity in retinas lacking photoreceptors by inserting a microbial light-activated channel in retinal ganglion cells and demonstrated that the treated retina from previously completely blind mice responded to light and produced activity in the visual cortex (Bi et al., 2006).

More recently, we established physiological role of different types of GCAPs in regulation of light response (Peshenko et al., 2004; Makino et al., 2012). Over several years, my lab created and characterized new mouse models in which genes coding for different forms of GCAPs were selectively disabled. In that study, we demonstrated how GCAPs activated cGMP production by sequentially turning on the activities of two different forms of RetGC cyclase by different forms of GCAPs in order to provide the required speed of excitation and recovery (Makino et al., 2012; Olshevskaya et al., 2012).

Other important subjects we are addressing at present relate to the protein interactions that create the active versus inactive states of the complex between RetGC and its regulatory sensor GCAP. We want to know how the enzyme and the sensor protein recognize each other, because this is the process that becomes affected by various blinding mutations. For a number of years, we attempted to establish what makes GCAP recognize its target enzyme, RetGC, and vise versa. Just last year, we completed extensive mapping of GCAP, by painstakingly probing the entire surface of the protein molecule with single amino acid substitutions and testing their effects on GCAP ability to bind and activate RetGC. We analyzed more than a hundred mutations in the protein and pinpointed the part responsible for its connecting to the cyclase (Peshenko et al., 2014). Conversely, by mutating the target enzyme, we established a particular portion of the RetGC molecule to which different GCAPs bind (Peshenko et al., 2015). Furthermore, we have just created a series of complex chimera proteins, in which different parts of RetGC were mutated and identified some amino acid residues that help RetGC recognize GCAPs. The new finding can provide a new mechanistic explanation of how GCAPs regulate RetGC. Most importantly, a new mutation in that portion of RetGC was found in patients suffering from an early-onset hereditary blindness, Leber's congenital amaurosis (LCA) (Jacobson et al., 2013).

[Igor Peshenko, PhD, assistant professor] This and other mutations causing LCA were clinically and biochemically characterized in a recent study conducted in collaboration with the groups of Samuel Jacobson and Arthur Cideciyan at University of Pennsylvania and Edwin Stone at University of Iowa (Jacobson et al., 2013). The study showed that a number of mutations in different parts of RetGC found in LCA patients disabled the cyclase activity and regulation by GCAPs, but the disabled photoreceptors remained alive and largely preserved. Therefore, LCA caused by mutations in RetGC emerges as a plausible object for treatment using gene therapy, in which a normal RetGC gene is supplied to the diseased photoreceptors by infecting the retina with modified viral particles carrying the gene. This approach successfully improved vision in mice lacking normal RetGC - a study led by William Hauswirth and Shannon Boye at University of Florida to which our group at Salus contributed biochemical characterization of the treated retinas (Boye et al., 2013). Current focus of the laboratory remains on the molecular mechanisms of vision and creating new genetic models replicating human hereditary blindness, such as LCA and dominant cone-rod degeneration.

These studies will help better understand the cause of the disease and develop strategies for gene therapy. This research continues to be supported by grants from the National Institutes of Health (NIH) and Pennsylvania Department of Health.