Rahul M. Kohli, M.D., Ph.D.
Assistant Professor, Medicine and Biochemistry & Biophysics
Perelman School of Medicine
University of Pennsylvania
502B Johnson Pavilion
36th and Hamilton Walk
Philadelphia, PA 19104-6073
Kohli Lab Site
Our laboratory focuses on the enzymatic generation of genomic diversity. We
utilize a broad array of approaches, including biochemical characterization
of enzyme mechanisms, chemical synthesis of enzyme probes, and biological assays
spanning immunology and virology to study this central tactic in the constant
battle between our immune system and pathogens.
From the host immune perspective, the generation of genomic diversity is used
as both a defensive and an offensive weapon. On the one hand, host mutator
enzymes such as Activation-Induced Cytidine Deaminase (AID) seed diversity
in the adaptive immune system by introducing targeted mutations into the immunoglobulin
locus that result in high affinity antibodies (somatic hypermutation) or altered
isotypes (class switch recombination). Conversely, related deaminases of the
innate immune system can directly attack retroviral threats by garbling the
pathogen genome through mutation, as accomplished by the deaminase APOBEC3G,
which restricts infection with HIV. Immune mutator enzymes, however, also pose
a risk to the host, as overexpression or dysregulation have been associated
From the pathogen perspective, alteration in key antigenic determinants at
a rate that outpaces immune responses is a potent means for evasion. Further,
rapid mutation may allow for the development of resistance to antimicrobials.
Our research program aims to understand the enzymatic basis for diversity
generation in the immune system and pathogens. We further aim to harness these
diversity-generating systems for directed evolution and to chemically perturb
these pathways to impede pathogen escape or prevent the neoplastic transformations
that can result from genomic mutation.
Rotation students are welcome starting Fall 2010.
Projects of interest to the laboratory include:
- Decipher the molecular basis for deamination by AID/APOBEC enzymes
and perturb deaminase immunological functions. Members of the
AID/APOBEC family are linked by the ability to bind nucleic acids, but
distinguished from one another by targeting distinctions, at a global level
(host vs. pathogen genome) and at a local level (targeting distinct DNA
hotspots for deamination). We use a combination of chemical perturbation
of substrates with site-directed mutagenesis to decipher the molecular
determinants of deamination and targeting. We further posit that mechanism
based chemical probes will offer needed insight into polynucleotide deaminase
function and yield lead compounds for inhibition of potentially oncogenic
AID/APOBEC activity. We aim to translate biochemical insights into immunologic
and virologic studies that can reveal the enzymatic roles in HIV restriction,
antibody diversity and unwanted chromosomal translocations.
- Harness the enzymes of antibody diversity for directed evolution. The
generation of antibody diversity is the most powerful natural example of
protein evolution. In this process, the targeted introduction of DNA lesions
into the immunoglobulin locus is coupled to error-prone, rather than error-free,
repair. Our growing understanding of the pathway of antibody diversification
offers an opportunity to harness the power of these enzymes for directed
protein evolution. We aim to introduce the enzymatic machinery of B-cells
into E. coli to yield a robust system for diversification and selection
of proteins with new and improved functions. This system has the potential
to offer a means to continuous, inducible evolution, overcoming limitations
to existing methods for protein evolution.
- Target pathogen pathways that promote evolution and resistance. Pathogens
diversity allows for escape from immune pressures or resistance to antimicrobials.
Diversity can be introduced by multiple means in pathogens: error prone replication
by HIV reverse transcriptase, activation of the SOS pathways in bacteria
and antigenic variation via gene conversion in trypanosomes represent a few
remarkable examples. These pathways share a common thread of using DNA repair
or replication enzymes in error prone manners and either revealing preexisting
hidden genetic variations or newly introducing mutations. Targeting the enzymes
that allow for the emergence of pathogen diversity offers an appealing, novel
target to prevent the emergence of resistance and attenuate pathogen virulence.
We initially aim to characterize the enzymes involved in the SOS pathway
in Pseudomonas and develop small molecule inhibitors of these enzymes
for evaluation as anti-infective agents with the ability to prevent the emergence
of drug resistance.